Method of treating psoriatic arthritis with increased interval dosing of anti-IL12/23 antibody

ABSTRACT

A method of treating an IL-12/23-related disease in a patient using an increasing dosing interval, comprises increasing the dosing interval of IL-12/IL-23 antibody to a patient, wherein the antibody is administered initially and after 4 weeks, after 16 weeks and after 28 weeks, and increasing the dosing interval after 28 weeks to an increased interval, e.g., every 16, 20 or 24 weeks.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/471,790, filed 28 Mar. 2017, now issued as U.S. Pat. No. 10,765,724,which claims the benefit of priority of U.S. Provisional Application No.62/314,697, filed Mar. 29, 2016, the entire contents of which areincorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name, JBI5084USDIV1SEQLIST.txt, creation date of Jul. 27, 2020 andhaving a size of 8 Kb. The sequence listing submitted via EFS-Web ispart of the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention concerns methods for treating an IL-12/23-relateddisease with an antibody that binds human IL-12 and/or human IL-23proteins using specific dosing regimens. In particular, it relates toidentification of an increased dosing (or maintenance) interval forsubcutaneously administered anti-IL-12/23p40 antibody and specificpharmaceutical compositions of an antibody, e.g., ustekinumab, which aresafe and effective for administration to patients with anIL-12/23-related disease.

BACKGROUND OF THE INVENTION

Interleukin (IL)-12 is a secreted heterodimeric cytokine comprised of 2disulfide-linked glycosylated protein subunits, designated p35 and p40for their approximate molecular weights. IL-12 is produced primarily byantigen-presenting cells and drives cell-mediated immunity by binding toa two-chain receptor complex that is expressed on the surface of T cellsor natural killer (NK) cells. The IL-12 receptor beta-1 (IL-12Rβ1) chainbinds to the p40 subunit of IL-12, providing the primary interactionbetween IL-12 and its receptor. However, it is IL-12p35 ligation of thesecond receptor chain, IL-12Rβ2, that confers intracellular signaling(e.g. STAT4 phosphorylation) and activation of the receptor-bearing cell(Presky et al, 1996). IL-12 signaling concurrent with antigenpresentation is thought to invoke T cell differentiation towards the Thelper 1 (Th1) phenotype, characterized by interferon gamma (IFNγ)production (Trinchieri, 2003). Th1 cells are believed to promoteimmunity to some intracellular pathogens, generate complement-fixingantibody isotypes, and contribute to tumor immunosurveillance. Thus,IL-12 is thought to be a significant component to host defense immunemechanisms.

It was discovered that the p40 protein subunit of IL-12 can alsoassociate with a separate protein subunit, designated p19, to form anovel cytokine, IL-23 (Oppman et al, 2000). IL-23 also signals through atwo-chain receptor complex. Since the p40 subunit is shared betweenIL-12 and IL-23, it follows that the IL-12101 chain is also sharedbetween IL-12 and IL-23. However, it is the IL-23p19 ligation of thesecond component of the IL-23 receptor complex, IL-23R, that confersIL-23 specific intracellular signaling (e.g., STAT3 phosphorylation) andsubsequent IL-17 production by T cells (Parham et al, 2002; Aggarwal etal. 2003). Studies have demonstrated that the biological functions ofIL-23 are distinct from those of IL-12, despite the structuralsimilarity between the two cytokines (Langrish et al, 2005).

Abnormal regulation of IL-12 and Th1 cell populations has beenassociated with many immune-mediated diseases since neutralization ofIL-12 by antibodies is effective in treating animal models of psoriasis,multiple sclerosis (MS), rheumatoid arthritis, inflammatory boweldisease, insulin-dependent (type 1) diabetes mellitus, and uveitis(Leonard et al, 1995; Hong et al, 1999; Malfait et al, 1998; Davidson etal, 1998). However, since these studies targeted the shared p40 subunit,both IL-12 and IL-23 were neutralized in vivo. Therefore, it was unclearwhether IL-12 or IL-23 was mediating disease, or if both cytokinesneeded to be inhibited to achieve disease suppression. Additionalstudies have confirmed through IL-23p19 deficient mice or specificantibody neutralization of IL-23 that IL-23 inhibition can provideequivalent benefit as anti-IL-12p40 strategies (Cua et al, 2003, Murphyet al, 2003, Benson et al 2004). Therefore, there is evidence for theroles of IL-12 and IL-23 in immune-mediated disease

Psoriasis is a chronic immune-mediated skin disorder with significantco-morbidities, such as psoriatic arthritis (PsA), depression,cardiovascular disease, hypertension, obesity, diabetes, metabolicsyndrome, and Crohn's disease. Plaque psoriasis is the most common formof the disease and manifests in well demarcated erythematous lesionstopped with white silver scales. Plaques are pruritic, painful and oftendisfiguring and a significant proportion of psoriatic patients haveplaques on hands/nails face, feet and genitalia. As such, psoriasis canimpose physical and psychosocial burdens that extend beyond the physicaldermatological symptoms and interfere with everyday activities. Forexample, psoriasis negatively impacts familial, spousal, social, andwork relationships, and is associated with a higher incidence ofdepression and increased suicidal tendencies.

Histologic characterization of psoriasis lesions reveals a thickenedepidermis resulting from aberrant keratinocyte proliferation anddifferentiation as well as dermal infiltration and co-localization ofCD3+T lymphocytes and dendritic cells. While the etiology of psoriasisis not well defined, gene and protein analysis have shown that IL-12,IL-23 and their downstream molecules are over-expressed in psoriaticlesions, and some may correlate with psoriasis disease severity. Sometherapies used in the treatment of psoriasis modulate IL-12 and IL-23levels, which is speculated to contribute to their efficacy. Th1 andTh17 cells can produce effector cytokines that induce the production ofvasodilators, chemoattractants and expression of adhesion molecules onendothelial cells which in turn, promote monocyte and neutrophilrecruitment, T cell infiltration, neovascularization and keratinocyteactivation and hyperplasia. Activated keratinocytes can producechemoattractant factors that promote neutrophil, monocyte, T cell, anddendritic cell trafficking, thus establishing a cycle of inflammationand keratinocyte hyperproliferation.

Results of three phase 3 clinical studies of the IL-12/23 antibodyustekinumab in the treatment of moderate-to-severe plaque psoriasis havebeen published. Ustekinumab administered by subcutaneous injection atweeks 0 and 4 and then once every 12 weeks exhibited rapid and sustainedclinical response, as assessed by the Psoriasis Area and Severity Index,a validated efficacy tool for psoriasis. A Phase 3 study comparingustekinumab with etanercept, a TNF antagonist, demonstrated that theefficacy of ustekinumab was superior to that of etanercept over a12-week period in patients with moderate-to-severe psoriasis. In twophase 3 clinical studies, Phoenix I and Phoenix II, ustekinumabexhibited a half life of approximately 3 weeks. Immune response ratesagainst ustekinumab ranged from 3 to 5%. In addition, reported adverseevents were relatively mild, with the majority of events includingsusceptibility to mild infections such as nasopharyngitis and upperrespiratory tract infection. Rates of infection were not higher inustekinumab-treated patients when compared with placebo-treated patientsover 12 weeks of therapy; nor were they increased in association withhigher, relative to lower, ustekinumab doses. Also, rates of seriousinfections, cardiovascular events, injection site reactions, andmalignancies were low. Taken together, the clinical observations ofustekinumab in psoriasis have supported its first-in-class status andconfirmed the fundamental role of IL-12 and/or IL-23 in psoriasispathogenesis.

SUMMARY OF THE INVENTION

In a first aspect, the invention concerns a method of treating anIL-12/23-related disease in a patient comprising subcutaneouslyadministering an anti-IL-12 and/or anti-IL-23 antibody, e.g., ananti-IL-12/23p40 (IL-12/23p40) antibody, to the patient, wherein theanti-IL-12/23p40 antibody is administered at an initial dose, a dose 4weeks thereafter, and at a dosing interval of once every 12 weeks andthat interval (the maintenance interval) is increased 28 weeks after theinitial dose. The increased dosing interval can be a set interval orcustomized based on when a patient experiences reappearance of thedisease state after withdrawal of or increased interval foradministration of the antibody therapy, e.g., in psoriasis a change inPGA and/or PASI scores. In one embodiment, after 28 weeks the dosinginterval is increased from once every 12 weeks to once every 16, 20 or24 weeks at 45 mg or 90 mg doses.

In an embodiment, the IL-12/23-related disease is selected from thegroup consisting of psoriasis, psoriatic arthritis, lupus, diabetes,Crohn's disease, ulcerative colitis and other inflammatory boweldiseases, sarcoidosis, ankylosing spondylitis (AS) and axialspondyloarthritis (nrAxSpA). In a preferred embodiment, theIL-12/23-related disease is psoriasis. In another embodiment, theIL-12/23-related disease is psoriatic arthritis.

The invention also concerns a method of treating psoriasis in a patientcomprising subcutaneously administering the anti-IL-12/23p40 antibodyustekinumab (Stelara®) to the patient, wherein the ustekinumab isadministered initially, 4 weeks after the initial dose, at a dosinginterval of once every 12 weeks until 28 weeks after an initial dose andthen is administered once every 16, 20 or 24 weeks.

In addition, the composition used in the method of the inventioncomprises a pharmaceutical composition comprising: an anti-IL-12/23p40antibody in an amount from about 1.0 μg/ml to about 1000 mg/ml,specifically a 45 mg or 90 mg dose. In a preferred embodiment theanti-IL-12/23p40 antibody is ustekinumab (Stelara®). In anotherembodiment, the pharmaceutical composition comprises an isolatedanti-IL-12/IL-23p40 antibody that binds a peptide chain comprisingresidues 1-88 of SEQ ID NO: 9; from about 0.27 to about 0.80 mgL-histidine per ml of the pharmaceutical composition; from about 0.69 toabout 2.1 mg L-histidine monohydrochloride monohydrate per ml of thepharmaceutical composition; from about 0.02 to about 0.06 mg polysorbate80 per ml of the pharmaceutical composition; and from about 65 to about87 mg of sucrose per ml of the pharmaceutical composition; wherein thediluent is water at standard state.

In another aspect of the invention the pharmaceutical compositioncomprises an isolated anti-IL-12/IL-23p40 antibody having (i) the heavychain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ IDNO: 3; and (ii) the light chain CDR amino acid sequences of SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO: 6; from about 0.27 to about 0.80 mgL-histidine per ml of the pharmaceutical composition; from about 0.69 toabout 2.1 mg L-histidine monohydrochloride monohydrate per ml of thepharmaceutical composition; from about 0.02 to about 0.06 mg polysorbate80 per ml of the pharmaceutical composition; and from about 65 to about87 mg of sucrose per ml of the pharmaceutical composition; wherein thediluent is water at standard state.

Another aspect of the method of the invention comprises administering apharmaceutical composition comprising an isolated anti-IL-12/IL-23p40antibody having the heavy chain variable region amino acid sequence ofSEQ ID NO: 7 and the light chain variable region amino acid sequence ofSEQ ID NO: 8; from about 0.27 to about 0.80 mg L-histidine per ml of thepharmaceutical composition; from about 0.69 to about 2.1 mg L-histidinemonohydrochloride monohydrate per ml of the pharmaceutical composition;from about 0.02 to about 0.06 mg polysorbate 80 per ml of thepharmaceutical composition; and from about 65 to about 87 mg of sucroseper ml of the pharmaceutical composition; wherein the diluent is waterat standard state.

Another aspect of the method is administering a pharmaceuticalcomposition comprising an isolated anti-IL-12/IL-23p40 antibody having(i) the heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3; and (ii) the light chain CDR amino acid sequencesof SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; about 0.53 mgL-histidine per ml of the pharmaceutical composition; about 1.37 mgL-histidine monohydrochloride monohydrate per ml of the pharmaceuticalcomposition; about 0.04 mg polysorbate 80 per ml of the pharmaceuticalcomposition; and about 76 mg of sucrose per ml of the pharmaceuticalcomposition; wherein the diluent is water at standard state.

A further aspect of the method is administering a pharmaceuticalcomposition comprising an isolated anti-IL-12/IL-23p40 antibody havingthe heavy chain variable region amino acid sequence of SEQ ID NO: 7 andthe light chain variable region amino acid sequence of SEQ ID NO: 8wherein the isolated antibody binds a peptide chain comprising residues1-88 of SEQ ID NO: 9; about 0.53 mg L-histidine per ml of thepharmaceutical composition; about 1.37 mg L-histidine monohydrochloridemonohydrate per ml of the pharmaceutical composition; about 0.04 mgpolysorbate 80 per ml of the pharmaceutical composition; and about 76 mgof sucrose per ml of the pharmaceutical composition; wherein the diluentis water at standard state.

In another aspect of the method is administering a pharmaceuticalcomposition comprising a binding compound that competes for binding withthe above-described antibodies, optionally at residues 1-88 of SEQ IDNO: 9; from about 0.27 to about 0.80 mg L-histidine per ml of thepharmaceutical composition; from about 0.69 to about 2.1 mg L-histidinemonohydrochloride monohydrate per ml of the pharmaceutical composition;from about 0.02 to about 0.06 mg polysorbate 80 per ml of thepharmaceutical composition; and from about 65 to about 87 mg of sucroseper ml of the pharmaceutical composition; wherein the diluent is waterat standard state.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the molecular structure of the bound complex of IL-12/p40Fab in a ribbon representation.

FIG. 2 shows the p40 mAb binding site (epitope) represented on themolecular surface in surface and ribbons representations. The D1 domainand Fv are isolated out of the complex structure for clarity. For the D1domain of p40, the molecular surface is shown. The Fv part of the Fab isshown in ribbons. Left panel: view down the antibody binding site, i.e.,the epitope. Middle panel: view ˜90° from view in left panel. Rightpanel: Ribbon representation of the residues of the epitope.

FIG. 3 shows the results of an ELISA evaluation of the IL-12 p40antibody bound to various p40 single mutants.

FIG. 4 shows the relative binding affinity of the p40 mAb to differentp40 muteins.

FIG. 5 shows a Study Schema of Ustekinumab in a Phase 3b, randomized,double-blind, active treatment-controlled, multicenter study with a4-week screening period, an open-label run-in period from Week 0 to Week28, a double-blind treatment period from Weeks 28 to Week 104, apost-treatment period through 116, and a safety follow-up via contact bytelephone or an onsite visit at Week 124.

FIG. 6 shows the percent of subjects achieving a PGA Score of Cleared(0) or Minimal (1) from Week 28 through Week 112 by Visit in StudyCNTO1275PS03009.

FIG. 7 shows percent of subjects achieving a PASI 75 Response from Week28 through Week 112 by Visit in Study CNTO1275PS03009.

FIG. 8 shows PGA responses of cleared (0) over time from Week 28 throughWeek 112.

FIG. 9 shows the percent of subjects achieving a PASI 90 Response fromWeek 28 through Week 112 by Visit in Study CNTO1275PS03009.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein the method of treatment of psoriasis comprisesadministering isolated, recombinant and/or synthetic anti-IL-12, IL-23and IL12/23p40 human antibodies and diagnostic and therapeuticcompositions, methods and devices.

As used herein, an “anti-IL-12 antibody,” “anti-IL-23 antibody,”“anti-IL-12/23p40 antibody,” “IL-12/23p40 antibody,” “antibody portion,”or “antibody fragment” and/or “antibody variant” and the like includeany protein or peptide containing molecule that comprises at least aportion of an immunoglobulin molecule, such as but not limited to, atleast one complementarity determining region (CDR) of a heavy or lightchain or a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework region, or any portion thereof, or at least one portion of anIL-12 and/or IL-23 receptor or binding protein, which can beincorporated into an antibody of the present invention. Such antibodyoptionally further affects a specific ligand, such as but not limitedto, where such antibody modulates, decreases, increases, antagonizes,agonizes, mitigates, alleviates, blocks, inhibits, abrogates and/orinterferes with at least one IL-12/23 activity or binding, or withIL-12/23 receptor activity or binding, in vitro, in situ and/or in vivo.As a non-limiting example, a suitable anti-IL-12/23p40 antibody,specified portion or variant of the present invention can bind at leastone IL-12/23 molecule, or specified portions, variants or domainsthereof. A suitable anti-IL-12/23p40 antibody, specified portion, orvariant can also optionally affect at least one of IL-12/23 activity orfunction, such as but not limited to, RNA, DNA or protein synthesis,IL-12/23 release, IL-12/23 receptor signaling, membrane IL-12/23cleavage, IL-12/23 activity, IL-12/23 production and/or synthesis.

The term “antibody” is further intended to encompass antibodies,digestion fragments, specified portions and variants thereof, includingantibody mimetics or comprising portions of antibodies that mimic thestructure and/or function of an antibody or specified fragment orportion thereof, including single chain antibodies and fragmentsthereof. Functional fragments include antigen-binding fragments thatbind to a mammalian IL-12/23. For example, antibody fragments capable ofbinding to IL-12/23 or portions thereof, including, but not limited to,Fab (e.g., by papain digestion), Fab′ (e.g., by pepsin digestion andpartial reduction) and F(ab′)2 (e.g., by pepsin digestion), facb (e.g.,by plasmin digestion), pFc′ (e.g., by pepsin or plasmin digestion), Fd(e.g., by pepsin digestion, partial reduction and reaggregation), Fv orscFv (e.g., by molecular biology techniques) fragments, are encompassedby the invention (see, e.g., Colligan, Immunology, supra).

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the C_(H)1 domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, C_(L),C_(H) domains (e.g., C_(H)1, C_(H)2, C_(H)3), hinge, (V_(L), V_(H))) issubstantially non-immunogenic in humans, with only minor sequencechanges or variations. A “human antibody” may also be an antibody thatis derived from or closely matches human germline immunoglobulinsequences. Human antibodies may include amino acid residues not encodedby germline immunoglobulin sequences (e.g., mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo). Often, this means that the human antibody is substantiallynon-immunogenic in humans. Human antibodies have been classified intogroupings based on their amino acid sequence similarities. Accordingly,using a sequence similarity search, an antibody with a similar linearsequence can be chosen as a template to create a human antibody.Similarly, antibodies designated primate (monkey, baboon, chimpanzee,etc.), rodent (mouse, rat, rabbit, guinea pig, hamster, and the like)and other mammals designate such species, sub-genus, genus, sub-family,and family specific antibodies. Further, chimeric antibodies can includeany combination of the above. Such changes or variations optionally andpreferably retain or reduce the immunogenicity in humans or otherspecies relative to non-modified antibodies. Thus, a human antibody isdistinct from a chimeric or humanized antibody.

It is pointed out that a human antibody can be produced by a non-humananimal or prokaryotic or eukaryotic cell that is capable of expressingfunctionally rearranged human immunoglobulin (e.g., heavy chain and/orlight chain) genes. Further, when a human antibody is a single chainantibody, it can comprise a linker peptide that is not found in nativehuman antibodies. For example, an Fv can comprise a linker peptide, suchas two to about eight glycine or other amino acid residues, whichconnects the variable region of the heavy chain and the variable regionof the light chain. Such linker peptides are considered to be of humanorigin.

Bispecific, heterospecific, heteroconjugate or similar antibodies canalso be used that are monoclonal, preferably, human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forat least one IL-12/23 protein, the other one is for any other antigen.Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986),each entirely incorporated herein by reference.

Anti-IL-12/23p40 antibodies (also termed IL-12/23p40 antibodies) (orantibodies to IL-23) useful in the methods and compositions of thepresent invention can optionally be characterized by high affinitybinding to IL-12/23p40 (or to IL-23) and, optionally and preferably,having low toxicity. In particular, an antibody, specified fragment orvariant of the invention, where the individual components, such as thevariable region, constant region and framework, individually and/orcollectively, optionally and preferably possess low immunogenicity, isuseful in the present invention. The antibodies that can be used in theinvention are optionally characterized by their ability to treatpatients for extended periods with measurable alleviation of symptomsand low and/or acceptable toxicity. Low or acceptable immunogenicityand/or high affinity, as well as other suitable properties, cancontribute to the therapeutic results achieved. “Low immunogenicity” isdefined herein as raising significant HAHA, HACA or HAMA responses inless than about 75%, or preferably less than about 50% of the patientstreated and/or raising low titres in the patient treated (less thanabout 300, preferably less than about 100 measured with a double antigenenzyme immunoassay) (Elliott et al., Lancet 344:1125-1127 (1994),entirely incorporated herein by reference). “Low immunogenicity” canalso be defined as the incidence of titrable levels of antibodies to theanti-IL-12 antibody in patients treated with anti-IL-12 antibody asoccurring in less than 25% of patients treated, preferably, in less than10% of patients treated with the recommended dose for the recommendedcourse of therapy during the treatment period.

Utility

The isolated nucleic acids of the present invention can be used forproduction of at least one anti-IL-12/23p40 (or anti-IL-23) antibody orspecified variant thereof, which can be used to measure or effect in ancell, tissue, organ or animal (including mammals and humans), todiagnose, monitor, modulate, treat, alleviate, help prevent theincidence of, or reduce the symptoms of, at least one IL-12/23condition, selected from, but not limited to, at least one of an immunedisorder or disease, a cardiovascular disorder or disease, aninfectious, malignant, and/or neurologic disorder or disease, or otherknown or specified IL-12/23 related condition.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least oneanti-IL-12/23p40 (or anti-IL-23) antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment, alleviation,prevention, or reduction in symptoms, effects or mechanisms. Theeffective amount can comprise an amount of about 0.001 to 500 mg/kg persingle (e.g., bolus), multiple or continuous administration, or toachieve a serum concentration of 0.01-5000 μg/ml serum concentration persingle, multiple, or continuous administration, or any effective rangeor value therein, as done and determined using known methods, asdescribed herein or known in the relevant arts.

Citations

All publications or patents cited herein, whether or not specificallydesignated, are entirely incorporated herein by reference as they showthe state of the art at the time of the present invention and/or toprovide description and enablement of the present invention.Publications refer to any scientific or patent publications, or anyother information available in any media format, including all recorded,electronic or printed formats. The following references are entirelyincorporated herein by reference: Ausubel, et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY(1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane,antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989);Colligan, et al., eds., Current Protocols in Immunology, John Wiley &Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols inProtein Science, John Wiley & Sons, NY, NY, (1997-2001).

Antibodies of the Present Invention—Production and Generation

At least one anti-IL-12/23p40 (or anti-IL-23) antibody used in themethod of the present invention can be optionally produced by a cellline, a mixed cell line, an immortalized cell or clonal population ofimmortalized cells, as well known in the art. See, e.g., Ausubel, etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001),each entirely incorporated herein by reference.

A preferred anti-IL-12/23p40 antibody is ustekinumab (Stelara®) havingthe heavy chain variable region amino acid sequence of SEQ ID NO: 7 andthe light chain variable region amino acid sequence of SEQ ID NO: 8 andhaving the heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ IDNO: 2, and SEQ ID NO: 3; and the light chain CDR amino acid sequences ofSEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. A preferred anti-IL-23antibody (binding specifically to IL-23 and not IL-12) is guselkumab(also referred to as CNTO1959 that comprises the variable regionsequences of SEQ ID NOS:106 and 116 in U.S. Pat. No. 7,935,344, theentire contents of which are incorporated herein by reference) and otherantibodies described in U.S. Pat. No. 7,935,344.

Human antibodies that are specific for human IL-12/23p40 or IL-23proteins or fragments thereof can be raised against an appropriateimmunogenic antigen, such as an isolated IL-12/23p40 protein, IL-23protein and/or a portion thereof (including synthetic molecules, such assynthetic peptides). Other specific or general mammalian antibodies canbe similarly raised. Preparation of immunogenic antigens, and monoclonalantibody production can be performed using any suitable technique.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line, such as, but not limited to,Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, L243, P3X63Ag8.653, Sp2SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1,JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMALWA, NEURO2A, or the like, or heteromylomas, fusion products thereof, or any cellor fusion cell derived therefrom, or any other suitable cell line asknown in the art) (see, e.g., www.atcc.org, www.lifetech.com., and thelike), with antibody producing cells, such as, but not limited to,isolated or cloned spleen, peripheral blood, lymph, tonsil, or otherimmune or B cell containing cells, or any other cells expressing heavyor light chain constant or variable or framework or CDR sequences,either as endogenous or heterologous nucleic acid, as recombinant orendogenous, viral, bacterial, algal, prokaryotic, amphibian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

Antibody producing cells can also be obtained from the peripheral bloodor, preferably, the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof, of the present invention. The fused cells (hybridomas)or recombinant cells can be isolated using selective culture conditionsor other suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;Biolnvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260 (May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; (Scripps); WO96/13583, WO97/08320(MorphoSys); WO95/16027 (BioInvent); WO88/06630; WO90/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, predecessor of Applied Molecular Evolution (AME), eachentirely incorporated herein by reference)) or that rely uponimmunization of transgenic animals (e.g., SCID mice, Nguyen et al.,Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev.Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998),each entirely incorporated by reference as well as related patents andapplications) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al.,Proc. Natl. Acad. Sci. USA, 95:14130-14135 (November 1998)); single cellantibody producing technologies (e.g., selected lymphocyte antibodymethod (“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al., J. Immunol.17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA93:7843-7848 (1996)); gel microdroplet and flow cytometry (Powell etal., Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, Mass.;Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al.,Bio/Technol. 13:787-790 (1995)); B-cell selection (Steenbakkers et al.,Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress Biotech,Vol. 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed.,Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcethat is non-human, e.g., but not limited to, mouse, rat, rabbit,non-human primate or other mammal. These non-human amino acid residuesare replaced by residues often referred to as “import” residues, whichare typically taken from an “import” variable, constant or other domainof a known human sequence.

Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast;www.atcc.org/phage/hdb.html; www.mrc-cpe.cam.ac.uk/ALIGNMENTS.php;www.kabatdatabase.com/top.html; ftp.ncbi.nih.gov/repository/kabat;www.sciquest.com; www.abcam.com;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/˜pedro/research_tools.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.hhmi.org/grants/lectures/1996/vlab;www.path.cam.ac.uk/˜mrc7/mikeimages.html;mcb.harvard.edu/BioLinks/Immunology.html; www.immunologylink.com;pathbox.wustl.edu/˜hcenter/index.html; www.appliedbiosystems.com;www.nal.usda.gov/awic/pubs/antibody;www.m.ehime-u.ac.jp/˜yasuhito/Elisa.html; www.biodesign.com;www.cancerresearchuk.org; www.biotech.ufl.edu; www.isac-net.org;baserv.uci.kun.nl/˜jraats/linksl.html;www.recab.uni-hd.de/immuno.bme.nwu.edu; www.mrc-cpe.cam.ac.uk;www.ibt.unam.mx/vir/V_mice.html; www.bioinforg.uk/abs;antibody.bath.ac.uk; www.unizh.ch; www.cryst.bbk.ac.uk/˜ubcg07s;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html;www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html; www.jerini.de; Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. Accordingly, partor all of the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions may be replacedwith human or other amino acids.

Antibodies can also optionally be humanized or human antibodiesengineered with retention of high affinity for the antigen and otherfavorable biological properties. To achieve this goal, humanized (orhuman) antibodies can be optionally prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, framework (FR) residuescan be selected and combined from the consensus and import sequences sothat the desired antibody characteristic, such as increased affinity forthe target antigen(s), is achieved.

In addition, the human IL-12/23p40 (or anti-IL-23) antibody used in themethod of the present invention may comprise a human germline lightchain framework. In particular embodiments, the light chain germlinesequence is selected from human VK sequences including, but not limitedto, A1, A10, A11, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30,A5, A7, B2, B3, L1, L10, L11, L12, L14, L15, L16, L18, L19, L2, L20,L22, L23, L24, L25, L4/18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2,O4, and 08. In certain embodiments, this light chain human germlineframework is selected from V1-11, V1-13, V1-16, V1-17, V1-18, V1-19,V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1, V2-11, V2-13,V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4, V4-1,V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6.

In other embodiments, the human IL-12/23p40 (or anti-IL-23) antibodyused in the method of the present invention may comprise a humangermline heavy chain framework. In particular embodiments, this heavychain human germline framework is selected from VH1-18, VH1-2, VH1-24,VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70,VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33,VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7,VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4,VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81.

In particular embodiments, the light chain variable region and/or heavychain variable region comprises a framework region or at least a portionof a framework region (e.g., containing 2 or 3 subregions, such as FR2and FR3). In certain embodiments, at least FRL1, FRL2, FRL3, or FRL4 isfully human. In other embodiments, at least FRH1, FRH2, FRH3, or FRH4 isfully human. In some embodiments, at least FRL1, FRL2, FRL3, or FRL4 isa germline sequence (e.g., human germline) or comprises human consensussequences for the particular framework (readily available at the sourcesof known human Ig sequences described above). In other embodiments, atleast FRH1, FRH2, FRH3, or FRH4 is a germline sequence (e.g., humangermline) or comprises human consensus sequences for the particularframework. In preferred embodiments, the framework region is a fullyhuman framework region.

Humanization or engineering of antibodies of the present invention canbe performed using any known method, such as but not limited to thosedescribed in, Winter (Jones et al., Nature 321:522 (1986); Riechmann etal., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)),Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol.Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A.89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat.Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192,5,723,323, 5,766886, 5714352, 6204023, 6180370, 5693762, 5530101,5585089, 5225539; 4816567, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, each entirely incorporated herein byreference, included references cited therein.

In certain embodiments, the antibody comprises an altered (e.g.,mutated) Fc region. For example, in some embodiments, the Fc region hasbeen altered to reduce or enhance the effector functions of theantibody. In some embodiments, the Fc region is an isotype selected fromIgM, IgA, IgG, IgE, or other isotype. Alternatively or additionally, itmay be useful to combine amino acid modifications with one or morefurther amino acid modifications that alter C1q binding and/or thecomplement dependent cytotoxicity function of the Fc region of an IL-12binding molecule. The starting polypeptide of particular interest may beone that binds to C1q and displays complement dependent cytotoxicity(CDC). Polypeptides with pre-existing C1q binding activity, optionallyfurther having the ability to mediate CDC may be modified such that oneor both of these activities are enhanced. Amino acid modifications thatalter C1q and/or modify its complement dependent cytotoxicity functionare described, for example, in WO0042072, which is hereby incorporatedby reference.

As disclosed above, one can design an Fc region of the human IL-12/23p40(or anti-IL-23) antibody of the present invention with altered effectorfunction, e.g., by modifying C1q binding and/or FcγR binding and therebychanging complement dependent cytotoxicity (CDC) activity and/orantibody-dependent cell-mediated cytotoxicity (ADCC) activity. “Effectorfunctions” are responsible for activating or diminishing a biologicalactivity (e.g., in a subject). Examples of effector functions include,but are not limited to: C1q binding; CDC; Fc receptor binding; ADCC;phagocytosis; down regulation of cell surface receptors (e.g., B cellreceptor; BCR), etc. Such effector functions may require the Fc regionto be combined with a binding domain (e.g., an antibody variable domain)and can be assessed using various assays (e.g., Fc binding assays, ADCCassays, CDC assays, etc.).

For example, one can generate a variant Fc region of the humanIL-12/23p40 (or anti-IL-23) antibody with improved C1q binding andimproved FcγRIIIbinding (e.g., having both improved ADCC activity andimproved CDC activity). Alternatively, if it is desired that effectorfunction be reduced or ablated, a variant Fc region can be engineeredwith reduced CDC activity and/or reduced ADCC activity. In otherembodiments, only one of these activities may be increased, and,optionally, also the other activity reduced (e.g., to generate an Fcregion variant with improved ADCC activity, but reduced CDC activity andvice versa).

Fc mutations can also be introduced in engineer to alter theirinteraction with the neonatal Fc receptor (FcRn) and improve theirpharmacokinetic properties. A collection of human Fc variants withimproved binding to the FcRn have been described (Shields et al.,(2001). High resolution mapping of the binding site on human IgG1 forFcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants withimproved binding to the FcγR, J. Biol. Chem. 276:6591-6604).

Another type of amino acid substitution serves to alter theglycosylation pattern of the Fc region of the human IL-12/23p40 (oranti-IL-23) antibody. Glycosylation of an Fc region is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. The recognition sequences forenzymatic attachment of the carbohydrate moiety to the asparagine sidechain peptide sequences are asparagine-χ-serine andasparagine-X-threonine, where X is any amino acid except proline. Thus,the presence of either of these peptide sequences in a polypeptidecreates a potential glycosylation site.

The glycosylation pattern may be altered, for example, by deleting oneor more glycosylation site(s) found in the polypeptide, and/or addingone or more glycosylation sites that are not present in the polypeptide.Addition of glycosylation sites to the Fc region of a human IL-12/23p40(or anti-IL-23) antibody is conveniently accomplished by altering theamino acid sequence such that it contains one or more of theabove-described tripeptide sequences (for N-linked glycosylation sites).An exemplary glycosylation variant has an amino acid substitution ofresidue Asn 297 of the heavy chain. The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the sequence of the original polypeptide (for O-linkedglycosylation sites). Additionally, a change of Asn 297 to Ala canremove one of the glycosylation sites.

In certain embodiments, the human IL-12/23p40 (or anti-IL-23) antibodyof the present invention is expressed in cells that express beta(1,4)-N-acetylglucosaminyltransferase III (GnT III), such that GnT IIIadds GlcNAc to the human IL-12 antibody. Methods for producingantibodies in such a fashion are provided in WO/9954342, WO/03011878,patent publication 20030003097A1, and Umana et al., NatureBiotechnology, 17:176-180, February 1999; all of which are hereinspecifically incorporated by reference in their entireties.

The anti-IL-12/23p40 (or anti-IL-23) antibody can also be optionallygenerated by immunization of a transgenic animal (e.g., mouse, rat,hamster, non-human primate, and the like) capable of producing arepertoire of human antibodies, as described herein and/or as known inthe art. Cells that produce a human anti-IL-12/23p40 (or anti-IL-23)antibody can be isolated from such animals and immortalized usingsuitable methods, such as the methods described herein.

Transgenic mice that can produce a repertoire of human antibodies thatbind to human antigens can be produced by known methods (e.g., but notlimited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126,5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al.;Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg etal. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151 B1,Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438 474B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440 A,Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol.6(4)579-591 (1994), Green et al, Nature Genetics 7:13-21 (1994), Mendezet al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic AcidsResearch 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad SciUSA 90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93(1995) and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), whichare each entirely incorporated herein by reference). Generally, thesemice comprise at least one transgene comprising DNA from at least onehuman immunoglobulin locus that is functionally rearranged, or which canundergo functional rearrangement. The endogenous immunoglobulin loci insuch mice can be disrupted or deleted to eliminate the capacity of theanimal to produce antibodies encoded by endogenous genes.

Screening antibodies for specific binding to similar proteins orfragments can be conveniently achieved using peptide display libraries.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. Antibodyscreening of peptide display libraries is well known in the art. Thedisplayed peptide sequences can be from 3 to 5000 or more amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence. Such methods are described in PCT Patent PublicationNos. 91/17271, 91/18980, 91/19818, and 93/08278.

Other systems for generating libraries of peptides have aspects of bothin vitro chemical synthesis and recombinant methods. See, PCT PatentPublication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Pat.Nos. 5,658,754; and 5,643,768. Peptide display libraries, vector, andscreening kits are commercially available from such suppliers asInvitrogen (Carlsbad, Calif.), and Cambridge antibody Technologies(Cambridgeshire, UK). See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666,4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730,5,763,733, 5,767,260, 5,856,456, assigned to Enzon; U.S. Pat. Nos.5,223,409, 5,403,484, 5,571,698, 5,837,500, assigned to Dyax, 5427908,5580717, assigned to Affymax; 5885793, assigned to Cambridge antibodyTechnologies; 5750373, assigned to Genentech, 5618920, 5595898, 5576195,5698435, 5693493, 5698417, assigned to Xoma, Colligan, supra; Ausubel,supra; or Sambrook, supra, each of the above patents and publicationsentirely incorporated herein by reference.

Antibodies used in the method of the present invention can also beprepared using at least one anti-IL-12/23p40 (or anti-IL-23) antibodyencoding nucleic acid to provide transgenic animals or mammals, such asgoats, cows, horses, sheep, rabbits, and the like, that produce suchantibodies in their milk. Such animals can be provided using knownmethods. See, e.g., but not limited to, U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, andthe like, each of which is entirely incorporated herein by reference.

Antibodies used in the method of the present invention can additionallybe prepared using at least one anti-IL-12/23p40 (or anti-IL-23) antibodyencoding nucleic acid to provide transgenic plants and cultured plantcells (e.g., but not limited to, tobacco and maize) that produce suchantibodies, specified portions or variants in the plant parts or incells cultured therefrom. As a non-limiting example, transgenic tobaccoleaves expressing recombinant proteins have been successfully used toprovide large amounts of recombinant proteins, e.g., using an induciblepromoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol.240:95-118 (1999) and references cited therein. Also, transgenic maizehave been used to express mammalian proteins at commercial productionlevels, with biological activities equivalent to those produced in otherrecombinant systems or purified from natural sources. See, e.g., Hood etal., Adv. Exp. Med. Biol. 464:127-147 (1999) and references citedtherein. Antibodies have also been produced in large amounts fromtransgenic plant seeds including antibody fragments, such as singlechain antibodies (scFv's), including tobacco seeds and potato tubers.See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) andreferences cited therein. Thus, antibodies of the present invention canalso be produced using transgenic plants, according to known methods.See also, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108(October, 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma etal., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc.Trans. 22:940-944 (1994); and references cited therein. Each of theabove references is entirely incorporated herein by reference.

The antibodies used in the method of the invention can bind humanIL-12/23p40 (or IL-23) with a wide range of affinities (K_(D)). In apreferred embodiment, a human mAb can optionally bind human IL-12/23p40(or IL-23) with high affinity. For example, a human mAb can bind humanIL-12/23p40 (or IL-23) with a K_(D) equal to or less than about 10⁻⁷ M,such as but not limited to, 0.1-9.9 (or any range or valuetherein)×10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ or any range orvalue therein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

Nucleic Acid Molecules

Using the information provided herein, for example, the nucleotidesequences encoding at least 70-100% of the contiguous amino acids of atleast one of the light or heavy chain variable or CDR regions of SEQ IDNOS: 1, 2, 3, 4, 5, 6, 7, 8, among other sequences disclosed herein,specified fragments, variants or consensus sequences thereof, or adeposited vector comprising at least one of these sequences, a nucleicacid molecule of the present invention encoding at least one anti-IL-12antibody can be obtained using methods described herein or as known inthe art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules used in the method of the presentinvention can include nucleic acid molecules comprising an open readingframe (ORF), optionally, with one or more introns, e.g., but not limitedto, at least one specified portion of at least one CDR, such as CDR1,CDR2 and/or CDR3 of at least one heavy chain (e.g., SEQ ID NOS:1-3) orlight chain (e.g., SEQ ID NOS:4-6); nucleic acid molecules comprisingthe coding sequence for an anti-IL-12/23p40 antibody or variable region(e.g., light and heavy chain variable regions of SEQ ID NOS:7 and 8);and nucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least oneanti-IL-12/23p40 (or anti-IL-23) antibody as described herein and/or asknown in the art. Of course, the genetic code is well known in the art.Thus, it would be routine for one skilled in the art to generate suchdegenerate nucleic acid variants that code for specific anti-IL-12/23p40(or anti-IL-23) antibodies used in the method of the present invention.See, e.g., Ausubel, et al., supra, and such nucleic acid variants areincluded in the present invention. Non-limiting examples of isolatednucleic acid molecules include nucleic acids encoding HC CDR1, HC CDR2,HC CDR3, LC CDR1, LC CDR2, and LC CDR3, respectively.

As indicated herein, nucleic acid molecules which comprise a nucleicacid encoding an anti-IL-12/23p40 (or anti-IL-23) antibody can include,but are not limited to, those encoding the amino acid sequence of anantibody fragment, by itself; the coding sequence for the entireantibody or a portion thereof; the coding sequence for an antibody,fragment or portion, as well as additional sequences, such as the codingsequence of at least one signal leader or fusion peptide, with orwithout the aforementioned additional coding sequences, such as at leastone intron, together with additional, non-coding sequences, includingbut not limited to, non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription,mRNA processing, including splicing and polyadenylation signals (forexample, ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding anantibody can be fused to a marker sequence, such as a sequence encodinga peptide that facilitates purification of the fused antibody comprisingan antibody fragment or portion.

Polynucleotides Selectively Hybridizing to a Polynucleotide as DescribedHerein

The method of the present invention uses isolated nucleic acids thathybridize under selective hybridization conditions to a polynucleotidedisclosed herein. Thus, the polynucleotides of this embodiment can beused for isolating, detecting, and/or quantifying nucleic acidscomprising such polynucleotides. For example, polynucleotides of thepresent invention can be used to identify, isolate, or amplify partialor full-length clones in a deposited library. In some embodiments, thepolynucleotides are genomic or cDNA sequences isolated, or otherwisecomplementary to, a cDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably, at least 85% or 90% full-length sequences, and,more preferably, at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides will encode at least a portion of anantibody. The polynucleotides embrace nucleic acid sequences that can beemployed for selective hybridization to a polynucleotide encoding anantibody of the present invention. See, e.g., Ausubel, supra; Colligan,supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids

The isolated nucleic acids can be made using (a) recombinant methods,(b) synthetic techniques, (c) purification techniques, and/or (d)combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention, excluding the coding sequence, is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra)

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions, such as RNA, cDNA, genomic DNA,or any combination thereof, can be obtained from biological sourcesusing any number of cloning methodologies known to those of skill in theart. In some embodiments, oligonucleotide probes that selectivelyhybridize, under stringent conditions, to the polynucleotides of thepresent invention are used to identify the desired sequence in a cDNA orgenomic DNA library. The isolation of RNA, and construction of cDNA andgenomic libraries, are well known to those of ordinary skill in the art.(See, e.g., Ausubel, supra; or Sambrook, supra)

Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide used in the method of the presentinvention, such as those disclosed herein. Probes can be used tohybridize with genomic DNA or cDNA sequences to isolate homologous genesin the same or different organisms. Those of skill in the art willappreciate that various degrees of stringency of hybridization can beemployed in the assay; and either the hybridization or the wash mediumcan be stringent. As the conditions for hybridization become morestringent, there must be a greater degree of complementarity between theprobe and the target for duplex formation to occur. The degree ofstringency can be controlled by one or more of temperature, ionicstrength, pH and the presence of a partially denaturing solvent, such asformamide. For example, the stringency of hybridization is convenientlyvaried by changing the polarity of the reactant solution through, forexample, manipulation of the concentration of formamide within the rangeof 0% to 50%. The degree of complementarity (sequence identity) requiredfor detectable binding will vary in accordance with the stringency ofthe hybridization medium and/or wash medium. The degree ofcomplementarity will optimally be 100%, or 70-100%, or any range orvalue therein. However, it should be understood that minor sequencevariations in the probes and primers can be compensated for by reducingthe stringency of the hybridization and/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; U.S. Pat.No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson, et al.; U.S.Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 to Gyllensten, etal; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No.4,656,134 to Ringold) and RNA mediated amplification that usesanti-sense RNA to the target sequence as a template for double-strandedDNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with thetradename NASBA), the entire contents of which references areincorporated herein by reference. (See, e.g., Ausubel, supra; orSambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides used in the method of thepresent invention and related genes directly from genomic DNA or cDNAlibraries. PCR and other in vitro amplification methods can also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of the desired mRNA in samples, for nucleic acidsequencing, or for other purposes. Examples of techniques sufficient todirect persons of skill through in vitro amplification methods are foundin Berger, supra, Sambrook, supra, and Ausubel, supra, as well asMullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCRProtocols A Guide to Methods and Applications, Eds., Academic PressInc., San Diego, Calif. (1990). Commercially available kits for genomicPCR amplification are known in the art. See, e.g., Advantage-GC GenomicPCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids used in the method of the present inventioncan also be prepared by direct chemical synthesis by known methods (see,e.g., Ausubel, et al., supra). Chemical synthesis generally produces asingle-stranded oligonucleotide, which can be converted intodouble-stranded DNA by hybridization with a complementary sequence, orby polymerization with a DNA polymerase using the single strand as atemplate. One of skill in the art will recognize that while chemicalsynthesis of DNA can be limited to sequences of about 100 or more bases,longer sequences can be obtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention uses recombinant expression cassettes comprising anucleic acid. A nucleic acid sequence, for example, a cDNA or a genomicsequence encoding an antibody used in the method of the presentinvention, can be used to construct a recombinant expression cassettethat can be introduced into at least one desired host cell. Arecombinant expression cassette will typically comprise a polynucleotideoperably linked to transcriptional initiation regulatory sequences thatwill direct the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in the intron) of a non-heterologousform of a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide. For example, endogenouspromoters can be altered in vivo or in vitro by mutation, deletionand/or substitution.

Vectors and Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules, host cells that are genetically engineered withthe recombinant vectors, and the production of at least oneanti-IL-12/23p40 (or anti-IL-23) antibody by recombinant techniques, asis well known in the art. See, e.g., Sambrook, et al., supra; Ausubel,et al., supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but are not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one antibody used in the method of the present invention can beexpressed in a modified form, such as a fusion protein, and can includenot only secretion signals, but also additional heterologous functionalregions. For instance, a region of additional amino acids, particularlycharged amino acids, can be added to the N-terminus of an antibody toimprove stability and persistence in the host cell, during purification,or during subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein used in the method of the present invention. Alternatively,nucleic acids can be expressed in a host cell by turning on (bymanipulation) in a host cell that contains endogenous DNA encoding anantibody. Such methods are well known in the art, e.g., as described inU.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirelyincorporated herein by reference.

Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653,SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va. (www.atcc.org). Preferred host cells include cells of lymphoidorigin, such as myeloma and lymphoma cells. Particularly preferred hostcells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) andSP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a particularlypreferred embodiment, the recombinant cell is a P3X63Ab8.653 or aSP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to, anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of an Antibody

An anti-IL-12/23-p40 (or anti-IL-23) antibody can be recovered andpurified from recombinant cell cultures by well-known methods including,but not limited to, protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Antibodies used in the method of the present invention include naturallypurified products, products of chemical synthetic procedures, andproducts produced by recombinant techniques from a eukaryotic host,including, for example, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the antibody can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

Anti-IL-12/IL-23p40 Antibodies.

An anti-IL-12/23p40 (or anti-IL-23) antibody according to the presentinvention includes any protein or peptide containing molecule thatcomprises at least a portion of an immunoglobulin molecule, such as butnot limited to, at least one ligand binding portion (LBP), such as butnot limited to, a complementarity determining region (CDR) of a heavy orlight chain or a ligand binding portion thereof, a heavy chain or lightchain variable region, a framework region (e.g., FR1, FR2, FR3, FR4 orfragment thereof, further optionally comprising at least onesubstitution, insertion or deletion), a heavy chain or light chainconstant region, (e.g., comprising at least one CH1, hinge1, hinge2,hinge3, hinge4, CH2, or CH3 or fragment thereof, further optionallycomprising at least one substitution, insertion or deletion), or anyportion thereof, that can be incorporated into an antibody. An antibodycan include or be derived from any mammal, such as but not limited to, ahuman, a mouse, a rabbit, a rat, a rodent, a primate, or any combinationthereof, and the like.

The isolated antibodies used in the method of the present inventioncomprise the antibody amino acid sequences disclosed herein encoded byany suitable polynucleotide, or any isolated or prepared antibody.Preferably, the human antibody or antigen-binding fragment binds humanIL-12/23 or IL-23 and, thereby, partially or substantially neutralizesat least one biological activity of the protein. An antibody, orspecified portion or variant thereof, that partially or preferablysubstantially neutralizes at least one biological activity of at leastone IL-12 or IL-23 protein or fragment can bind the protein or fragmentand thereby inhibit activities mediated through the binding of IL-12 orIL-23 to the IL-12 or IL-23 receptor or through other IL-12-dependent ormediated mechanisms. As used herein, the term “neutralizing antibody”refers to an antibody that can inhibit an IL-12 or IL-23-dependentactivity by about 20-120%, preferably by at least about 10, 20, 30, 40,50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100% or more depending on the assay. The capacity of an anti-IL-12/23p40antibody to inhibit an IL-12/23-dependent activity is preferablyassessed by at least one suitable IL-12/23 protein or receptor assay, asdescribed herein and/or as known in the art. A human antibody can be ofany class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise akappa or lambda light chain. In one embodiment, the human antibodycomprises an IgG heavy chain or defined fragment, for example, at leastone of isotypes, IgG1, IgG2, IgG3 or IgG4 (e.g., γ1, γ2, γ3, γ4).Antibodies of this type can be prepared by employing a transgenic mouseor other trangenic non-human mammal comprising at least one human lightchain (e.g., IgG, IgA, and IgM) transgenes as described herein and/or asknown in the art. In another embodiment, the anti-human IL-12/23p40 (oranti-IL-23) human antibody comprises an IgG1 heavy chain and an IgG1light chain.

An antibody binds at least one specified epitope specific to at leastone IL-12/23 protein, subunit, fragment, portion or any combinationthereof. The at least one epitope can comprise at least one antibodybinding region that comprises at least one portion of the protein, whichepitope is preferably comprised of at least one extracellular, soluble,hydrophillic, external or cytoplasmic portion of the protein. The atleast one specified epitope can comprise any combination of at least oneamino acid sequence of at least 1-3 amino acids to the entire specifiedportion of contiguous amino acids of SEQ ID NO:9, for example, aminoacid residues 15, 17-21, 23, 40-43, 45-47, 54-56 and 58-62.

Generally, the human antibody or antigen-binding fragment will comprisean antigen-binding region that comprises at least one humancomplementarity determining region (CDR1, CDR2 and CDR3) or variant ofat least one heavy chain variable region and at least one humancomplementarity determining region (CDR1, CDR2 and CDR3) or variant ofat least one light chain variable region. The CDR sequences may bederived from human germline sequences or closely match the germlinesequences. For example, the CDRs from a synthetic library derived fromthe original non-human CDRs can be used. These CDRs may be formed byincorporation of conservative substitutions from the original non-humansequence. As a non-limiting example, the antibody or antigen-bindingportion or variant can comprise at least one of the heavy chain CDR3having an amino acid sequence selected from the group consisting of SEQID NOS:1-3, and/or a light chain CDR3 having an amino acid sequenceselected from the group consisting of SEQ ID NOS:4-6. In a particularembodiment, the antibody or antigen-binding fragment can have anantigen-binding region that comprises at least a portion of at least oneheavy chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acidsequence of the corresponding CDRs 1, 2, and/or 3 (e.g., SEQ ID NOS:1,2, and/or 3). In another particular embodiment, the antibody orantigen-binding portion or variant can have an antigen-binding regionthat comprises at least a portion of at least one light chain CDR (i.e.,CDR1, CDR2 and/or CDR3) having the amino acid sequence of thecorresponding CDRs 1, 2 and/or 3.

Such antibodies can be prepared by chemically joining together thevarious portions (e.g., CDRs, framework) of the antibody usingconventional techniques, by preparing and expressing a (i.e., one ormore) nucleic acid molecule that encodes the antibody using conventionaltechniques of recombinant DNA technology or by using any other suitablemethod.

The anti-IL-12/23p40 (or anti-IL-23) antibody can comprise at least oneof a heavy or light chain variable region having a defined amino acidsequence. For example, in a preferred embodiment, the anti-IL-12/23p40antibody comprises at least one of at least one heavy chain variableregion, optionally having the amino acid sequence of SEQ ID NO:7 and/orat least one light chain variable region, optionally having the aminoacid sequence of SEQ ID NO:8. Antibodies that bind to human IL-12/23 andthat comprise a defined heavy or light chain variable region can beprepared using suitable methods, such as phage display (Katsube, Y., etal., Int J Mol. Med, 1(5):863-868 (1998)) or methods that employtransgenic animals, as known in the art and/or as described herein. Forexample, a transgenic mouse, comprising a functionally rearranged humanimmunoglobulin heavy chain transgene and a transgene comprising DNA froma human immunoglobulin light chain locus that can undergo functionalrearrangement, can be immunized with human IL-12/23 or a fragmentthereof to elicit the production of antibodies. If desired, the antibodyproducing cells can be isolated and hybridomas or other immortalizedantibody-producing cells can be prepared as described herein and/or asknown in the art. Alternatively, the antibody, specified portion orvariant can be expressed using the encoding nucleic acid or portionthereof in a suitable host cell.

The invention also relates to antibodies, antigen-binding fragments,immunoglobulin chains and CDRs comprising amino acids in a sequence thatis substantially the same as an amino acid sequence described herein.Preferably, such antibodies or antigen-binding fragments and antibodiescomprising such chains or CDRs can bind human IL-12/23 with highaffinity (e.g., K_(D) less than or equal to about 10⁻⁹M). Amino acidsequences that are substantially the same as the sequences describedherein include sequences comprising conservative amino acidsubstitutions, as well as amino acid deletions and/or insertions. Aconservative amino acid substitution refers to the replacement of afirst amino acid by a second amino acid that has chemical and/orphysical properties (e.g., charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include, without limitation,replacement of one amino acid by another within the following groups:lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate(E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine(Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine(I), proline (P), phenylalanine (F), tryptophan (W), methionine (M),cysteine (C) and glycine (G); F, W and Y; C, S and T.

Amino Acid Codes

The amino acids that make up anti-IL-12/23p40 (or anti-IL-23) antibodiesof the present invention are often abbreviated. The amino aciddesignations can be indicated by designating the amino acid by itssingle letter code, its three letter code, name, or three nucleotidecodon(s) as is well understood in the art (see Alberts, B., et al.,Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., NewYork, 1994):

SINGLE THREE THREE LETTER LETTER NUCLEOTIDE CODE CODE NAME CODON(S) AAla Alanine GCA, GCC, GCG, GCU C Cys Cysteine UGC, UGU D Asp Asparticacid GAC, GAU E Glu Glutamic acid GAA, GAG F Phe Phenylanine UUC, UUU GGly Glycine GGA, GGC, GGG, GGU H His Histidine CAC, CAU I Ile IsoleucineAUA, AUC, AUU K Lys Lysine AAA, AAG L Leu Leucine UUA, UUG, CUA, CUC,CUG, CUU M Met Methionine AUG N Asn Asparagine AAC, AAU P Pro ProlineCCA, CCC, CCG, CCU Q Gln Glutamine CAA, CAG R Arg Arginine AGA, AGG,CGA, CGC, CGG, CGU S Ser Serine AGC, AGU, UCA, UCC, UCG, UCU T ThrThreonine ACA, ACC, ACG, ACU V Val Valine GUA, GUC, GUG, GUU W TrpTryptophan UGG Y Tyr Tyrosine UAC, UAUAn anti-IL-12/23p40 (or anti-IL-23) antibody used in the method of thepresent invention can include one or more amino acid substitutions,deletions or additions, either from natural mutations or humanmanipulation, as specified herein.

The number of amino acid substitutions a skilled artisan would makedepends on many factors, including those described above. Generallyspeaking, the number of amino acid substitutions, insertions ordeletions for any given anti-IL-12/23p40 (or anti-IL-23) antibody,fragment or variant will not be more than 40, 30, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or anyrange or value therein, as specified herein.

Amino acids in an anti-IL-12/23p40 (or anti-IL-23) antibody that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g.,Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science244:1081-1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to, at least one IL-12 or IL-23 neutralizing activity. Sitesthat are critical for antibody binding can also be identified bystructural analysis, such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904(1992) and de Vos, et al., Science 255:306-312 (1992)). The residues onthe IL-12123p40 antibody involved in IL-12 binding have been identifiedbased upon the co-crystal structure of the IL-12/23p40 antibody andIL-12 p40 antigen. These are shown in Table 5 below.

Anti-IL-12/23p40 antibodies can include, but are not limited to, atleast one portion, sequence or combination selected from 5 to all of thecontiguous amino acids of at least one of SEQ ID NOS:1, 2, 3, 4, 5, 6.

IL-12/23p40 antibodies or specified portions or variants can include,but are not limited to, at least one portion, sequence or combinationselected from at least 3-5 contiguous amino acids of SEQ ID NO:1, 5-17contiguous amino acids of SEQ ID NO:2, 5-10 contiguous amino acids ofSEQ ID NO:3, 5-11 contiguous amino acids of SEQ ID NO:4, 5-7 contiguousamino acids of SEQ ID NO:5; 5-9 contiguous amino acids of SEQ ID NO:6;Leu21, Lys76, Met83, Ser85 of SEQ ID NO:7.

An anti-IL-12/23p40 antibody can further optionally comprise apolypeptide of at least one of 70-100% of 5, 17, 10, 11, 7, 9, 119, or108 contiguous amino acids of at least one of SEQ ID NOS:1, 2, 3, 4, 5,6, 7 or 8. In one embodiment, the amino acid sequence of animmunoglobulin chain, or portion thereof (e.g., variable region, CDR)has about 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 or any range or value therein) to the amino acidsequence of the corresponding chain of at least one of SEQ ID NOS: 1, 2,3, 4, 5, 6, 7 or 8. For example, the amino acid sequence of a lightchain variable region can be compared with the sequence of SEQ ID NOS:4, 5, 6, or 8, or the amino acid sequence of a heavy chain CDR3 can becompared with SEQ ID NOS: 1, 2, 3, or 7. Preferably, 70-100% amino acididentity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any rangeor value therein) is determined using a suitable computer algorithm, asknown in the art.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as determined by the match between strings of such sequences.“Identity” and “similarity” can be readily calculated by known methods,including, but not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., Siam J. Applied Math., 48:1073 (1988). In addition, values forpercentage identity can be obtained from amino acid and nucleotidesequence alignments generated using the default settings for the AlignXcomponent of Vector NTI Suite 8.0 (Informax, Frederick, Md.).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215:403-410 (1990)). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLMNIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410(1990). The well-known Smith Waterman algorithm may also be used todetermine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing: (1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-453(1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci, USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidesequence comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include thefollowing:

(1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid sequencecomparisons.

By way of example, a polynucleotide sequence may be identical to anothersequence, that is 100% identical, or it may include up to a certaininteger number of nucleotide alterations as compared to the referencesequence. Such alterations are selected from the group consisting of atleast one nucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein the alterations may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in the sequence by the numerical percent of the respectivepercent identity (divided by 100) and subtracting that product from thetotal number of nucleotides in the sequence, or:

n.sub.n.ltorsim.x.sub.n −(x.sub.n.y),

wherein n.sub.n is the number of nucleotide alterations, x.sub.n is thetotal number of nucleotides in sequence, and y is, for instance, 0.70for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, etc.,and wherein any non-integer product of x.sub.n and y is rounded down tothe nearest integer prior to subtracting from x.sub.n.

Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO: 7 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations. Similarly, a polypeptidesequence may be identical to the reference sequence of SEQ ID NO: 7,that is be 100% identical, or it may include up to a certain integernumber of amino acid alterations as compared to the reference sequencesuch that the percentage identity is less than 100%. Such alterationsare selected from the group consisting of at least one amino aciddeletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein the alterations may occur at theamino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO: 7 by thenumerical percent of the respective percent identity (divided by 100)and then subtracting that product from the total number of amino acidsin SEQ ID NO: 7, or:

n.sub.a.ltorsim.x.sub.a −(x.sub.a.y),

wherein n.sub.a is the number of amino acid alterations, x.sub.a is thetotal number of amino acids in SEQ ID NO: 7, and y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integerproduce of x.sub.a and y is rounded down to the nearest integer prior tosubtracting it from x.sub.a.

Exemplary heavy chain and light chain variable regions sequences andportions thereof are provided in SEQ ID NOS:1-8. The antibodies of thepresent invention, or specified variants thereof, can comprise anynumber of contiguous amino acid residues from an antibody of the presentinvention, wherein that number is selected from the group of integersconsisting of from 10-100% of the number of contiguous residues in ananti-IL-12 antibody. Optionally, this subsequence of contiguous aminoacids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 ormore amino acids in length, or any range or value therein. Further, thenumber of such subsequences can be any integer selected from the groupconsisting of from 1 to 20, such as at least 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and, preferably, at least 50%, 60%, or 70%, and, mostpreferably, at least 80%, 90%, or 95%-100% or more (including, withoutlimitation, up to 10 times the specific activity) of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity are well known to those of skill in the art.

In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments can comprise oneor more organic moieties that are covalently bonded, directly orindirectly, to the antibody. Each organic moiety that is bonded to anantibody or antigen-binding fragment of the invention can independentlybe a hydrophilic polymeric group, a fatty acid group or a fatty acidester group. As used herein, the term “fatty acid” encompassesmono-carboxylic acids and di-carboxylic acids. A “hydrophilic polymericgroup,” as the term is used herein, refers to an organic polymer that ismore soluble in water than in octane. For example, polylysine is moresoluble in water than in octane. Thus, an antibody modified by thecovalent attachment of polylysine is encompassed by the invention.Hydrophilic polymers suitable for modifying antibodies of the inventioncan be linear or branched and include, for example, polyalkane glycols(e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like),carbohydrates (e.g., dextran, cellulose, oligosaccharides,polysaccharides and the like), polymers of hydrophilic amino acids(e.g., polylysine, polyarginine, polyaspartate and the like), polyalkaneoxides (e.g., polyethylene oxide, polypropylene oxide and the like) andpolyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifiesthe antibody of the invention has a molecular weight of about 800 toabout 150,000 Daltons as a separate molecular entity. For example,PEG₅₀₀₀ and PEG_(20,000), wherein the subscript is the average molecularweight of the polymer in Daltons, can be used. The hydrophilic polymericgroup can be substituted with one to about six alkyl, fatty acid orfatty acid ester groups. Hydrophilic polymers that are substituted witha fatty acid or fatty acid ester group can be prepared by employingsuitable methods. For example, a polymer comprising an amine group canbe coupled to a carboxylate of the fatty acid or fatty acid ester, andan activated carboxylate (e.g., activated with N, N-carbonyldiimidazole) on a fatty acid or fatty acid ester can be coupled to ahydroxyl group on a polymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate (Cu, laurate),n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate),n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate),n-triacontanoate (C₃₀), n-tetracontanoate (C40), cis-49-octadecanoate(C18, oleate), all cis-45,8,11,14-eicosatetraenoate (C20, arachidonate),octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that comprise a linear or branchedlower alkyl group. The lower alkyl group can comprise from one to abouttwelve, preferably, one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups, such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example, adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom, such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate, as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221, the entire teachings of which areincorporated herein by reference.)

The modified antibodies can be produced by reacting a human antibody orantigen-binding fragment with a modifying agent. For example, theorganic moieties can be bonded to the antibody in a non-site specificmanner by employing an amine-reactive modifying agent, for example, anNHS ester of PEG. Modified human antibodies or antigen-binding fragmentscan also be prepared by reducing disulfide bonds (e.g., intra-chaindisulfide bonds) of an antibody or antigen-binding fragment. The reducedantibody or antigen-binding fragment can then be reacted with athiol-reactive modifying agent to produce the modified antibody of theinvention. Modified human antibodies and antigen-binding fragmentscomprising an organic moiety that is bonded to specific sites of anantibody of the present invention can be prepared using suitablemethods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem.,3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994);Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al.,Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol.Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson,G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif.(1996).

The method of the present invention also uses an anti-IL-12/23p40antibody composition comprising at least one, at least two, at leastthree, at least four, at least five, at least six or moreanti-IL-12/23p40 antibodies thereof, as described herein and/or as knownin the art that are provided in a non-naturally occurring composition,mixture or form. Such compositions comprise non-naturally occurringcompositions comprising at least one or two full length, C-and/orN-terminally deleted variants, domains, fragments, or specifiedvariants, of the anti-IL-12/23p40 antibody amino acid sequence selectedfrom the group consisting of 70-100% of the contiguous amino acids ofSEQ ID NOS: 7 or 8, or specified fragments, domains or variants thereof.Preferred anti-IL-12/23p40 antibody compositions include at least one ortwo full length, fragments, domains or variants as at least one CDR orLBP containing portions of the anti-IL-12/23p40 antibody sequencedescribed herein, for example, 70-100% of SEQ ID NOS:1-6, 7, or 8, orspecified fragments, domains or variants thereof. Further preferredcompositions comprise, for example, 40-99% of at least one of 70-100% ofSEQ ID NOS: 1-6, 7, or 8, etc., or specified fragments, domains orvariants thereof. Such composition percentages are by weight, volume,concentration, molarity, or molality as liquid or dry solutions,mixtures, suspension, emulsions, particles, powder, or colloids, asknown in the art or as described herein.

Antibody Compositions Comprising Further Therapeutically ActiveIngredients

The antibody compositions used in the method of the invention canoptionally further comprise an effective amount of at least one compoundor protein selected from at least one of an anti-infective drug, acardiovascular (CV) system drug, a central nervous system (CNS) drug, anautonomic nervous system (ANS) drug, a respiratory tract drug, agastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid orelectrolyte balance, a hematologic drug, an antineoplastic, animmunomodulation drug, an ophthalmic, otic or nasal drug, a topicaldrug, a nutritional drug or the like. Such drugs are well known in theart, including formulations, indications, dosing and administration foreach presented herein (see, e.g., Nursing 2001 Handbook of Drugs,21^(st) edition, Springhouse Corp., Springhouse, P A, 2001; HealthProfessional's Drug Guide 2001, ed., Shannon, Wilson, Stang,Prentice-Hall, Inc, Upper Saddle River, N.J.; Pharmcotherapy Handbook,Wells et al., ed., Appleton & Lange, Stamford, Conn., each entirelyincorporated herein by reference).

By way of example of the drugs that can be combined with the antibodiesfor the method of the present invention, the anti-infective drug can beat least one selected from amebicides or at least one antiprotozoals,anthelmintics, antifungals, antimalarials, antituberculotics or at leastone antileprotics, aminoglycosides, penicillins, cephalosporins,tetracyclines, sulfonamides, fluoroquinolones, antivirals, macrolideanti-infectives, and miscellaneous anti-infectives. The hormonal drugcan be at least one selected from corticosteroids, androgens or at leastone anabolic steroid, estrogen or at least one progestin, gonadotropin,antidiabetic drug or at least one glucagon, thyroid hormone, thyroidhormone antagonist, pituitary hormone, and parathyroid-like drug. The atleast one cephalosporin can be at least one selected from cefaclor,cefadroxil, cefazolin sodium, cefdinir, cefepime hydrochloride,cefixime, cefmetazole sodium, cefonicid sodium, cefoperazone sodium,cefotaxime sodium, cefotetan disodium, cefoxitin sodium, cefpodoximeproxetil, cefprozil, ceftazidime, ceftibuten, ceftizoxime sodium,ceftriaxone sodium, cefuroxime axetil, cefuroxime sodium, cephalexinhydrochloride, cephalexin monohydrate, cephradine, and loracarbef.

The at least one corticosteroid can be at least one selected frombetamethasone, betamethasone acetate or betamethasone sodium phosphate,betamethasone sodium phosphate, cortisone acetate, dexamethasone,dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisoneacetate, hydrocortisone, hydrocortisone acetate, hydrocortisonecypionate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, prednisolone, prednisolone acetate,prednisolone sodium phosphate, prednisolone tebutate, prednisone,triamcinolone, triamcinolone acetonide, and triamcinolone diacetate. Theat least one androgen or anabolic steroid can be at least one selectedfrom danazol, fluoxymesterone, methyltestosterone, nandrolone decanoate,nandrolone phenpropionate, testosterone, testosterone cypionate,testosterone enanthate, testosterone propionate, and testosteronetransdermal system.

The at least one immunosuppressant can be at least one selected fromazathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immuneglobulin, muromonab-CD3, mycophenolate mofetil, mycophenolate mofetilhydrochloride, sirolimus, and tacrolimus.

The at least one local anti-infective can be at least one selected fromacyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazolenitrate, clindamycin phosphate, clotrimazole, econazole nitrate,erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate,metronidazole (topical), miconazole nitrate, mupirocin, naftifinehydrochloride, neomycin sulfate, nitrofurazone, nystatin, silversulfadiazine, terbinafine hydrochloride, terconazole, tetracyclinehydrochloride, tioconazole, and tolnaftate. The at least one scabicideor pediculicide can be at least one selected from crotamiton, lindane,permethrin, and pyrethrins. The at least one topical corticosteroid canbe at least one selected from betamethasone dipropionate, betamethasonevalerate, clobetasol propionate, desonide, desoximetasone,dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate,fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasonepropionate, halcionide, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocortisone valerate, mometasone furoate,and triamcinolone acetonide. (See, e.g., pp. 1098-1136 of Nursing 2001Drug Handbook.)

Anti-IL-12/23p40 (or anti-IL-23) antibody compositions can furthercomprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneanti-IL-12/23p40 (or anti-IL-23) antibody contacted or administered to acell, tissue, organ, animal or patient in need of such modulation,treatment or therapy, optionally further comprising at least oneselected from at least one TNF antagonist (e.g., but not limited to aTNF chemical or protein antagonist, TNF monoclonal or polyclonalantibody or fragment, a soluble TNF receptor (e.g., p55, p70 or p85) orfragment, fusion polypeptides thereof, or a small molecule TNFantagonist, e.g., TNF binding protein I or II (TBP-1 or TBP-II),nerelimonmab, infliximab, eternacept, CDP-571, CDP-870, afelimomab,lenercept, and the like), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a cytokine or a cytokineantagonist. Non-limiting examples of such cytokines include, but are notlimited to, any of IL-1 to IL-23 et al. (e.g., IL-1, IL-2, etc.).Suitable dosages are well known in the art. See, e.g., Wells et al.,eds., Pharmacotherapy Handbook, 2^(nd) Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),each of which references are entirely incorporated herein by reference.

Anti-IL-12/23p40 antibody compounds, compositions or combinations usedin the method of the present invention can further comprise at least oneof any suitable auxiliary, such as, but not limited to, diluent, binder,stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvantor the like. Pharmaceutically acceptable auxiliaries are preferred.Non-limiting examples of, and methods of preparing such sterilesolutions are well known in the art, such as, but limited to, Gennaro,Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack PublishingCo. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers can beroutinely selected that are suitable for the mode of administration,solubility and/or stability of the anti-IL-12/23p40 antibody, fragmentor variant composition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include, but are not limited to, proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars, such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin, such ashuman serum albumin (HSA), recombinant human albumin (rHA), gelatin,casein, and the like. Representative amino acid/antibody components,which can also function in a buffering capacity, include alanine,glycine, arginine, betaine, histidine, glutamic acid, aspartic acid,cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, and the like. One preferred amino acid isglycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-IL-12/23p40 antibody (or anti-IL-23) compositions can also includea buffer or a pH adjusting agent; typically, the buffer is a saltprepared from an organic acid or base. Representative buffers includeorganic acid salts, such as salts of citric acid, ascorbic acid,gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid,or phthalic acid; Tris, tromethamine hydrochloride, or phosphatebuffers. Preferred buffers for use in the present compositions areorganic acid salts, such as citrate.

Additionally, anti-IL-12/23p40 (or anti-IL-23) antibody compositions caninclude polymeric excipients/additives, such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates, such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the anti-IL-12/23p40 (or anti-IL-23) antibody,portion or variant compositions according to the invention are known inthe art, e.g., as listed in “Remington: The Science & Practice ofPharmacy,” 19^(th) ed., Williams & Williams, (1995), and in the“Physician's Desk Reference,” 52^(nd) ed., Medical Economics, Montvale,N.J. (1998), the disclosures of which are entirely incorporated hereinby reference. Preferred carrier or excipient materials are carbohydrates(e.g., saccharides and alditols) and buffers (e.g., citrate) orpolymeric agents. An exemplary carrier molecule is themucopolysaccharide, hyaluronic acid, which may be useful forintraarticular delivery.

Formulations

As noted above, the invention provides for stable formulations, whichpreferably comprise a phosphate buffer with saline or a chosen salt, aswell as preserved solutions and formulations containing a preservativeas well as multi-use preserved formulations suitable for pharmaceuticalor veterinary use, comprising at least one anti-IL-12/23p40 (oranti-IL-23) antibody in a pharmaceutically acceptable formulation.Preserved formulations contain at least one known preservative oroptionally selected from the group consisting of at least one phenol,m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol,magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof inan aqueous diluent. Any suitable concentration or mixture can be used asknown in the art, such as 0.001-5%, or any range or value therein, suchas, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03,0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein.Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g.,0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9,1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01),0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0%alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075,0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9,1.0%), and the like.

As noted above, the method of the invention uses an article ofmanufacture, comprising packaging material and at least one vialcomprising a solution of at least one anti-IL-12/23p40 (or anti-IL-23)antibody with the prescribed buffers and/or preservatives, optionally inan aqueous diluent, wherein said packaging material comprises a labelthat indicates that such solution can be held over a period of 1, 2, 3,4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours orgreater. The invention further uses an article of manufacture,comprising packaging material, a first vial comprising lyophilizedanti-IL-12/23p40 (or anti-IL-23) antibody, and a second vial comprisingan aqueous diluent of prescribed buffer or preservative, wherein saidpackaging material comprises a label that instructs a patient toreconstitute the anti-IL-12/23p40 (or anti-IL-23) antibody in theaqueous diluent to form a solution that can be held over a period oftwenty-four hours or greater.

The anti-IL-12/23p40 (or anti-IL-23) antibody used in accordance withthe present invention can be produced by recombinant means, includingfrom mammalian cell or transgenic preparations, or can be purified fromother biological sources, as described herein or as known in the art.

The range of the anti-IL-12/23p40 (or anti-IL-23) antibody includesamounts yielding upon reconstitution, if in a wet/dry system,concentrations from about 1.0 μg/ml to about 1000 mg/ml, although lowerand higher concentrations are operable and are dependent on the intendeddelivery vehicle, e.g., solution formulations will differ fromtransdermal patch, pulmonary, transmucosal, or osmotic or micro pumpmethods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g., isotonicity agents, buffers, antioxidants, andpreservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably, the formulations of the presentinvention have a pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably, sodium phosphate,particularly, phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants, such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators, such asEDTA and EGTA, can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations can be prepared by a process which comprises mixing atleast one anti-IL-12/23p40 (or anti-IL-23) antibody and a preservativeselected from the group consisting of phenol, m-cresol, p-cresol,o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one anti-IL-12/23p40 (oranti-IL-23) antibody and preservative in an aqueous diluent is carriedout using conventional dissolution and mixing procedures. To prepare asuitable formulation, for example, a measured amount of at least oneanti-IL-12/23p40 (or anti-IL-23) antibody in buffered solution iscombined with the desired preservative in a buffered solution inquantities sufficient to provide the protein and preservative at thedesired concentrations. Variations of this process would be recognizedby one of ordinary skill in the art. For example, the order thecomponents are added, whether additional additives are used, thetemperature and pH at which the formulation is prepared, are all factorsthat can be optimized for the concentration and means of administrationused.

The formulations can be provided to patients as clear solutions or asdual vials comprising a vial of lyophilized anti-IL-12/23p40 (oranti-IL-23) antibody that is reconstituted with a second vial containingwater, a preservative and/or excipients, preferably, a phosphate bufferand/or saline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present articles of manufacture are useful for administration over aperiod ranging from immediate to twenty-four hours or greater.Accordingly, the presently claimed articles of manufacture offersignificant advantages to the patient. Formulations of the invention canoptionally be safely stored at temperatures of from about 2° C. to about40° C. and retain the biologically activity of the protein for extendedperiods of time, thus allowing a package label indicating that thesolution can be held and/or used over a period of 6, 12, 18, 24, 36, 48,72, or 96 hours or greater. If preserved diluent is used, such label caninclude use up to 1-12 months, one-half, one and a half, and/or twoyears.

The solutions of anti-IL-12/23p40 (or anti-IL-23) antibody can beprepared by a process that comprises mixing at least one antibody in anaqueous diluent. Mixing is carried out using conventional dissolutionand mixing procedures. To prepare a suitable diluent, for example, ameasured amount of at least one antibody in water or buffer is combinedin quantities sufficient to provide the protein and, optionally, apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least oneanti-IL-12/23p40 (or anti-IL-23) antibody that is reconstituted with asecond vial containing the aqueous diluent. Either a single solutionvial or dual vial requiring reconstitution can be reused multiple timesand can suffice for a single or multiple cycles of patient treatment andthus provides a more convenient treatment regimen than currentlyavailable.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneanti-IL-12/23p40 (or anti-IL-23) antibody that is reconstituted with asecond vial containing the aqueous diluent. The clear solution in thiscase can be up to one liter or even larger in size, providing a largereservoir from which smaller portions of the at least one antibodysolution can be retrieved one or multiple times for transfer intosmaller vials and provided by the pharmacy or clinic to their customersand/or patients.

Recognized devices comprising single vial systems include pen-injectordevices for delivery of a solution, such as BD Pens, BD Autojector®,Humaject®, NovoPen®, B-D®Pen, AutoPen®, and OptiPen®, GenotropinPen®,Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®,Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®, Smartject®e.g., as made or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com), and similarly suitable devices. Recognized devicescomprising a dual vial system include those pen-injector systems forreconstituting a lyophilized drug in a cartridge for delivery of thereconstituted solution, such as the HumatroPen®. Examples of otherdevices suitable include pre-filled syringes, auto-injectors, needlefree injectors, and needle free IV infusion sets.

The products may include packaging material. The packaging materialprovides, in addition to the information required by the regulatoryagencies, the conditions under which the product can be used. Thepackaging material of the present invention provides instructions to thepatient, as applicable, to reconstitute the at least oneanti-IL-12/23p40 (or anti-IL-23) antibody in the aqueous diluent to forma solution and to use the solution over a period of 2-24 hours orgreater for the two vial, wet/dry, product. For the single vial,solution product, pre-filled syringe or auto-injector, the labelindicates that such solution can be used over a period of 2-24 hours orgreater. The products are useful for human pharmaceutical product use.

The formulations used in the method of the present invention can beprepared by a process that comprises mixing an anti-IL-12/23p40 (oranti-IL-23) antibody and a selected buffer, preferably, a phosphatebuffer containing saline or a chosen salt. Mixing the anti-IL-12/23p40(or anti-IL-23) antibody and buffer in an aqueous diluent is carried outusing conventional dissolution and mixing procedures. To prepare asuitable formulation, for example, a measured amount of at least oneantibody in water or buffer is combined with the desired buffering agentin water in quantities sufficient to provide the protein and buffer atthe desired concentrations. Variations of this process would berecognized by one of ordinary skill in the art. For example, the orderthe components are added, whether additional additives are used, thetemperature and pH at which the formulation is prepared, are all factorsthat can be optimized for the concentration and means of administrationused.

The method of the invention provides pharmaceutical compositionscomprising various formulations useful and acceptable for administrationto a human or animal patient. Such pharmaceutical compositions areprepared using water at “standard state” as the diluent and routinemethods well known to those of ordinary skill in the art. For example,buffering components such as histidine and histidine monohydrochloridehydrate, may be provided first followed by the addition of anappropriate, non-final volume of water diluent, sucrose and polysorbate80 at “standard state.” Isolated antibody may then be added. Last, thevolume of the pharmaceutical composition is adjusted to the desiredfinal volume under “standard state” conditions using water as thediluent. Those skilled in the art will recognize a number of othermethods suitable for the preparation of the pharmaceutical compositions.

The pharmaceutical compositions may be aqueous solutions or suspensionscomprising the indicated mass of each constituent per unit of watervolume or having an indicated pH at “standard state.” As used herein,the term “standard state” means a temperature of 25° C. +/−2° C. and apressure of 1 atmosphere. The term “standard state” is not used in theart to refer to a single art recognized set of temperatures or pressure,but is instead a reference state that specifies temperatures andpressure to be used to describe a solution or suspension with aparticular composition under the reference “standard state” conditions.This is because the volume of a solution is, in part, a function oftemperature and pressure. Those skilled in the art will recognize thatpharmaceutical compositions equivalent to those disclosed here can beproduced at other temperatures and pressures. Whether suchpharmaceutical compositions are equivalent to those disclosed hereshould be determined under the “standard state” conditions defined above(e.g. 25° C. +/−2° C. and a pressure of 1 atmosphere).

Importantly, such pharmaceutical compositions may contain componentmasses “about” a certain value (e.g. “about 0.53 mg L-histidine”) perunit volume of the pharmaceutical composition or have pH values about acertain value. A component mass present in a pharmaceutical compositionor pH value is “about” a given numerical value if the isolated antibodypresent in the pharmaceutical composition is able to bind a peptidechain comprising residues 1-88 of SEQ ID NO: 9 while the isolatedantibody is present in the pharmaceutical composition or after theisolated antibody has been removed from the pharmaceutical composition(e.g., by dilution). Stated differently, a value, such as a componentmass value or pH value, is “about” a given numerical value when thebinding activity of the isolated antibody is maintained and detectableafter placing the isolated antibody in the pharmaceutical composition.

Competition binding analysis is performed to determine if theIL-12/23p40 (or anti-IL-23) mAbs bind to similar or different epitopesand/or compete with each other. Abs are individually coated on ELISAplates. Competing mAbs are added, followed by the addition ofbiotinylated hrIL-12/23p40. For positive control, the same mAb forcoating may be used as the competing mAb (“self-competition”).IL-12/23p40 binding is detected using streptavidin. These resultsdemonstrate whether the mAbs recognize similar or partially overlappingepitopes on IL-12/23p40.

One aspect of the method of the invention administers to a patient apharmaceutical composition comprising an isolated antibody that binds apeptide chain comprising residues 1-88 of SEQ ID NO: 9; from about 0.27to about 0.80 mg L-histidine per ml of the pharmaceutical composition;from about 0.69 to about 2.1 mg L-histidine monohydrochloridemonohydrate per ml of the pharmaceutical composition; from about 0.02 toabout 0.06 mg polysorbate 80 per ml of the pharmaceutical composition;and from about 65 to about 87 mg of sucrose per ml of the pharmaceuticalcomposition; wherein the diluent is water at standard state.

Another aspect of the invention comprises administering a pharmaceuticalcomposition comprising an isolated antibody having (i) the heavy chainCDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:3; and (ii) the light chain CDR amino acid sequences of SEQ ID NO: 4,SEQ ID NO: 5, and SEQ ID NO: 6 wherein the isolated antibody binds apeptide chain comprising residues 1-88 of SEQ ID NO: 9; from about 0.27to about 0.80 mg L-histidine per ml of the pharmaceutical composition;from about 0.69 to about 2.1 mg L-histidine monohydrochloridemonohydrate per ml of the pharmaceutical composition; from about 0.02 toabout 0.06 mg polysorbate 80 per ml of the pharmaceutical composition;and from about 65 to about 87 mg of sucrose per ml of the pharmaceuticalcomposition; wherein the diluent is water at standard state.

Another aspect of the method of the invention is administering to apatient a pharmaceutical composition comprising an isolatedanti-IL-12/23p40 antibody having the heavy chain amino acid sequence ofSEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8wherein the isolated antibody binds a peptide chain comprising residues1-88 of SEQ ID NO: 9; from about 0.27 to about 0.80 mg L-histidine perml of the pharmaceutical composition; from about 0.69 to about 2.1 mgL-histidine monohydrochloride monohydrate per ml of the pharmaceuticalcomposition; from about 0.02 to about 0.06 mg polysorbate 80 per ml ofthe pharmaceutical composition; and from about 65 to about 87 mg ofsucrose per ml of the pharmaceutical composition; wherein the diluent iswater at standard state.

In one embodiment of the pharmaceutical compositions, the isolatedantibody concentration is from about 77 to about 104 mg per ml of thepharmaceutical composition. In another embodiment of the pharmaceuticalcompositions the pH is from about 5.5 to about 6.5.

Another aspect of the method of the invention administers to a patient apharmaceutical composition comprising an isolated anti-IL-12/23p40antibody having (i) the heavy chain CDR amino acid sequences of SEQ IDNO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and (ii) the light chain CDRamino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6wherein the isolated antibody binds a peptide chain comprising residues1-88 of SEQ ID NO: 9; about 0.53 mg L-histidine per ml of thepharmaceutical composition; about 1.37 mg L-histidine monohydrochloridemonohydrate per ml of the pharmaceutical composition; about 0.04 mgpolysorbate 80 per ml of the pharmaceutical composition; and about 76 mgof sucrose per ml of the pharmaceutical composition; wherein the diluentis water at standard state.

A further aspect of the method of the invention administers to a patienta pharmaceutical composition comprising an isolated anti-IL-12/23p40antibody having the heavy chain amino acid sequence of SEQ ID NO: 7 andthe light chain amino acid sequence of SEQ ID NO: 8 wherein the isolatedantibody binds a peptide chain comprising residues 1-88 of SEQ ID NO: 9;about 0.53 mg L-histidine per ml of the pharmaceutical composition;about 1.37 mg L-histidine monohydrochloride monohydrate per ml of thepharmaceutical composition; about 0.04 mg polysorbate 80 per ml of thepharmaceutical composition; and about 76 mg of sucrose per ml of thepharmaceutical composition; wherein the diluent is water at standardstate.

In one embodiment, the isolated antibody concentration is about 90 mgper ml of the pharmaceutical composition. In another embodiment of thesepharmaceutical compositions the pH is about 6.0. Another aspect of theinvention is a method using a pharmaceutical composition comprising anantibody that competes for binding with an anti-IL-12/23p40 antibody asrecited herein, e.g., binds a peptide chain comprising residues 1-88 ofSEQ ID NO: 9; from about 0.27 to about 0.80 mg L-histidine per ml of thepharmaceutical composition; from about 0.69 to about 2.1 mg L-histidinemonohydrochloride monohydrate per ml of the pharmaceutical composition;from about 0.02 to about 0.06 mg polysorbate 80 per ml of thepharmaceutical composition; and from about 65 to about 87 mg of sucroseper ml of the pharmaceutical composition; wherein the diluent is waterat standard state.

The stable or preserved formulations can be provided to patients asclear solutions or as dual vials comprising a vial of lyophilized atleast one anti-IL-12/23p40 antibody that is reconstituted with a secondvial containing a preservative or buffer and excipients in an aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

Other formulations or methods of stabilizing the anti-IL-12/23p40 (oranti-IL-23) antibody may result in other than a clear solution oflyophilized powder comprising the antibody. Among non-clear solutionsare formulations comprising particulate suspensions, said particulatesbeing a composition containing the anti-IL-12/23p40 (or anti-IL-23)antibody in a structure of variable dimension and known variously as amicrosphere, microparticle, nanoparticle, nanosphere, or liposome. Suchrelatively homogenous, essentially spherical, particulate formulationscontaining an active agent can be formed by contacting an aqueous phasecontaining the active agent and a polymer and a nonaqueous phasefollowed by evaporation of the nonaqueous phase to cause the coalescenceof particles from the aqueous phase as taught in U.S. Pat. No.4,589,330. Porous microparticles can be prepared using a first phasecontaining active agent and a polymer dispersed in a continuous solventand removing said solvent from the suspension by freeze-drying ordilution-extraction-precipitation as taught in U.S. Pat. No. 4,818,542.Preferred polymers for such preparations are natural or syntheticcopolymers or polymers selected from the group consisting of gleatinagar, starch, arabinogalactan, albumin, collagen, polyglycolic acid,polylactic aced, glycolide-L(−) lactide poly(episilon-caprolactone,poly(epsilon-caprolactone-CO-lactic acid),poly(epsilon-caprolactone-CO-glycolic acid), poly(B-hydroxy butyricacid), polyethylene oxide, polyethylene, poly(alkyl-2-cyanoacrylate),poly(hydroxyethyl methacrylate), polyamides, poly(amino acids),poly(2-hydroxyethyl DL-aspartamide), poly(ester urea),poly(L-phenylalanine/ethylene glycol/1,6-diisocyanatohexane) andpoly(methyl methacrylate). Particularly preferred polymers arepolyesters, such as polyglycolic acid, polylactic aced, glycolide-L(−)lactide poly(episilon-caprolactone, poly(epsilon-caprolactone-CO-lacticacid), and poly(epsilon-caprolactone-CO-glycolic acid. Solvents usefulfor dissolving the polymer and/or the active include: water,hexafluoroisopropanol, methylenechloride, tetrahydrofuran, hexane,benzene, or hexafluoroacetone sesquihydrate. The process of dispersingthe active containing phase with a second phase may include pressureforcing said first phase through an orifice in a nozzle to affectdroplet formation.

Dry powder formulations may result from processes other thanlyophilization, such as by spray drying or solvent extraction byevaporation or by precipitation of a crystalline composition followed byone or more steps to remove aqueous or nonaqueous solvent. Preparationof a spray-dried antibody preparation is taught in U.S. Pat. No.6,019,968. The antibody-based dry powder compositions may be produced byspray drying solutions or slurries of the antibody and, optionally,excipients, in a solvent under conditions to provide a respirable drypowder. Solvents may include polar compounds, such as water and ethanol,which may be readily dried. Antibody stability may be enhanced byperforming the spray drying procedures in the absence of oxygen, such asunder a nitrogen blanket or by using nitrogen as the drying gas. Anotherrelatively dry formulation is a dispersion of a plurality of perforatedmicrostructures dispersed in a suspension medium that typicallycomprises a hydrofluoroalkane propellant as taught in WO 9916419. Thestabilized dispersions may be administered to the lung of a patientusing a metered dose inhaler. Equipment useful in the commercialmanufacture of spray dried medicaments are manufactured by Buchi Ltd. orNiro Corp.

An anti-IL-12/23p40 (or anti-IL-23) antibody in either the stable orpreserved formulations or solutions described herein, can beadministered to a patient in accordance with the present invention via avariety of delivery methods including SC or IM injection; transdermal,pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump,or other means appreciated by the skilled artisan, as well-known in theart.

Therapeutic Applications

The present invention also provides a method for modulating or treatingat least one IL-12/23 related disease, in a cell, tissue, organ, animal,or patient, as known in the art or as described herein, using at leastone IL-12/23p40 (or anti-IL-23) antibody of the present invention, e.g.,administering or contacting the cell, tissue, organ, animal, or patientwith a therapeutic effective amount of IL-12/23p40 (or anti-IL-23)antibody. The present invention also provides a method for modulating ortreating at least one IL-12/23 related disease, in a cell, tissue,organ, animal, or patient including, but not limited to, at least one ofobesity, an immune related disease, a cardiovascular disease, aninfectious disease, a malignant disease or a neurologic disease.

The present invention also provides a method for modulating or treatingat least one IL-12/23 related immune related disease, in a cell, tissue,organ, animal, or patient including, but not limited to, at least one ofpsoriasis, psoriatic arthritis, rheumatoid arthritis, juvenilerheumatoid arthritis, systemic onset juvenile rheumatoid arthritis,ankylosing spondilitis, gastric ulcer, seronegative arthropathies,osteoarthritis, osteolysis, aseptic loosening of orthopedic implants,inflammatory bowel disease, ulcerative colitis, systemic lupuserythematosus, antiphospholipid syndrome, iridocyclitis/uveitis/opticneuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitivity reactions, allergic rhinitis, hayfever, perennial rhinitis, conjunctivitis, endometriosis, asthma,urticaria, systemic anaphalaxis, dermatitis, pernicious anemia,hemolytic disease, thrombocytopenia, graft rejection of any organ ortissue, kidney transplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynaud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, POEMS syndrome (polyneuropathy,organomegaly, endocrinopathy, monoclonal gammopathy, and skin changessyndrome), polyneuropathy, organomegaly, endocrinopathy, monoclonalgammopathy, skin changes syndrome, antiphospholipid syndrome, pemphigus,scleroderma, mixed connective tissue disease, idiopathic Addison'sdisease, diabetes mellitus, chronic active hepatitis, primary billiarycirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, type IVhypersensitivity, contact dermatitis, hypersensitivity pneumonitis,allograft rejection, granulomas due to intracellular organisms, drugsensitivity, metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, alopecia, nephroticsyndrome, nephritis, glomerular nephritis, acute renal failure,hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy, anti-cd3therapy, cytokine therapy, chemotherapy, radiation therapy (e.g.,including but not limited to, asthenia, anemia, cachexia, and the like),chronic salicylate intoxication, and the like. See, e.g., the MerckManual, 12th-17th Editions, Merck & Company, Rahway, N.J. (1972, 1977,1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al., eds.,Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), eachentirely incorporated by reference. Such a method can optionallycomprise administering an effective amount of a composition orpharmaceutical composition comprising at least one TNF antibody orspecified portion or variant to a cell, tissue, organ, animal or patientin need of such modulation, treatment or therapy. See, e.g., the MerckManual, 16^(th) Edition, Merck & Company, Rahway, N.J. (1992).

The present invention also provides a method for modulating or treatingpsoriasis, psoriatic arthritis, Crohn's disease, other inflammatorybowel diseases, lupus, sarcoidosis, AS or nrAxSpA, among the otherdiseases listed above as IL-12/23 related, in a cell, tissue, organ,animal, or patient including, but not limited to, at least one of immunerelated disease, cardiovascular disease, infectious, malignant and/orneurologic disease. Such a method can optionally comprise administeringan effective amount of at least one composition or pharmaceuticalcomposition comprising an anti-IL-12/23p40 (or anti-IL-23) antibody to acell, tissue, organ, animal or patient in need of such modulation,treatment or therapy.

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising an anti-IL-12/23p40 (or anti-IL-23) antibody to a cell,tissue, organ, animal or patient in need of such modulation, treatmentor therapy. Such a method can optionally further compriseco-administration or combination therapy for treating such diseases ordisorders, wherein the administering of said at least oneanti-IL-12/23p40 (or anti-IL-23) antibody, specified portion or variantthereof, further comprises administering, before concurrently, and/orafter, at least one selected from at least one TNF antagonist (e.g., butnot limited to, a TNF chemical or protein antagonist, TNF monoclonal orpolyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70or p85) or fragment, fusion polypeptides thereof, or a small moleculeTNF antagonist, e.g., TNF binding protein I or II (TBP-1 or TBP-II),nerelimonmab, infliximab, eternacept (Enbrel™), adalimulab (Humira™)CDP-571, CDP-870, afelimomab, lenercept, and the like), an antirheumatic(e.g., methotrexate, auranofin, aurothioglucose, azathioprine, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropoietin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, C A (2000); Nursing 2001 Handbook of Drugs, 21^(st) edition,Springhouse Corp., Springhouse, P A, 2001; Health Professional's DrugGuide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, UpperSaddle River, N.J., each of which references are entirely incorporatedherein by reference.

Therapeutic Treatments

Typically, treatment of pathologic conditions is affected byadministering an effective amount or dosage of an anti-IL-12/23p40 (oranti-IL-23) antibody composition that total, on average, a range from atleast about 0.01 to 500 milligrams of an anti-IL-12/23p40 (oranti-IL-23) antibody per kilogram of patient per dose, and, preferably,from at least about 0.1 to 100 milligrams antibody/kilogram of patientper single or multiple administration, depending upon the specificactivity of the active agent contained in the composition.Alternatively, the effective serum concentration can comprise 0.1-5000μg/ml serum concentration per single or multiple administration.Suitable dosages are known to medical practitioners and will, of course,depend upon the particular disease state, specific activity of thecomposition being administered, and the particular patient undergoingtreatment. In some instances, to achieve the desired therapeutic amount,it can be necessary to provide for repeated administration, i.e.,repeated individual administrations of a particular monitored or metereddose, where the individual administrations are repeated until thedesired daily dose or effect is achieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0,5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14,14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9,19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and, preferably, 0.1 to10 milligrams per kilogram per administration or in sustained releaseform is effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or, alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or, alternatively or additionally, at least oneof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 years, or any combination thereof, using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.001 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion, particle, powder, or lyophilized powderin association, or separately provided, with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, dextrose solution, and 1-10% human serumalbumin. Liposomes and nonaqueous vehicles, such as fixed oils, can alsobe used. The vehicle or lyophilized powder can contain additives thatmaintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by known or suitable techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Alternative Administration

Many known and developed modes can be used according to the presentinvention for administering pharmaceutically effective amounts of ananti-IL-12/23p40 (or anti-IL-23) antibody. While pulmonaryadministration is used in the following description, other modes ofadministration can be used according to the present invention withsuitable results. IL-12/23p40 (or anti-IL-23) antibodies of the presentinvention can be delivered in a carrier, as a solution, emulsion,colloid, or suspension, or as a dry powder, using any of a variety ofdevices and methods suitable for administration by inhalation or othermodes described here within or known in the art.

Parenteral Formulations and Administration

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols, such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent, such as aqueous solution, asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthetic mono- or di- or tri-glycerides. Parental administration isknown in the art and includes, but is not limited to, conventional meansof injections, a gas pressured needle-less injection device as describedin U.S. Pat. No. 5,851,198, and a laser perforator device as describedin U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.

Alternative Delivery

The invention further relates to the administration of ananti-IL-12/23p40 (or anti-IL-23) antibody by parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal,or transdermal means. An anti-IL-12/23p40 (or anti-IL-23) antibodycomposition can be prepared for use for parenteral (subcutaneous,intramuscular or intravenous) or any other administration particularlyin the form of liquid solutions or suspensions; for use in vaginal orrectal administration particularly in semisolid forms, such as, but notlimited to, creams and suppositories; for buccal, or sublingualadministration, such as, but not limited to, in the form of tablets orcapsules; or intranasally, such as, but not limited to, the form ofpowders, nasal drops or aerosols or certain agents; or transdermally,such as not limited to a gel, ointment, lotion, suspension or patchdelivery system with chemical enhancers such as dimethyl sulfoxide toeither modify the skin structure or to increase the drug concentrationin the transdermal patch (Junginger, et al. In “Drug PermeationEnhancement;” Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. NewYork 1994, entirely incorporated herein by reference), or with oxidizingagents that enable the application of formulations containing proteinsand peptides onto the skin (WO 98/53847), or applications of electricfields to create transient transport pathways, such as electroporation,or to increase the mobility of charged drugs through the skin, such asiontophoresis, or application of ultrasound, such as sonophoresis (U.S.Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patentsbeing entirely incorporated herein by reference).

Having generally described the invention, the same will be more readilyunderstood by reference to the following Examples, which are provided byway of illustration and are not intended as limiting. Further details ofthe invention are illustrated by the following non-limiting Examples.The disclosures of all citations in the specification are expresslyincorporated herein by reference.

Example 1: Cloning and Expression of IL-12 Antibody in Mammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors, such as pIRES1neo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells. Alternatively, the gene can beexpressed in stable cell lines that contain the gene integrated into achromosome. The co-transfection with a selectable marker, such as dhfr,gpt, neomycin, or hygromycin, allows the identification and isolation ofthe transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition to the 3′ intron,the polyadenylation and termination signal of the rat preproinsulingene.

Cloning and Expression in CHO Cells.

The vector pC4 is used for the expression of IL-12/23p40 antibody.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (e.g., alpha minusMEM, Life Technologies, Gaithersburg, Md.) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., F. W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L.Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M.J. Page and M. A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grownin increasing concentrations of MTX develop resistance to the drug byoverproducing the target enzyme, DHFR, as a result of amplification ofthe DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach can be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained that contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

High efficiency promoters other than the strong promoter of the longterminal repeat (LTR) of the Rous Sarcoma Virus can also be used for theexpression, e.g., the human b-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems andsimilar systems can be used to express the IL-12 in a regulated way inmammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA89: 5547-5551 (1992)). For the polyadenylation of the mRNA, othersignals, e.g., from the human growth hormone or globin genes, can beused as well. Stable cell lines carrying a gene of interest integratedinto the chromosomes can also be selected upon co-transfection with aselectable marker, such as gpt, G418 or hygromycin. It is advantageousto use more than one selectable marker in the beginning, e.g., G418 plusmethotrexate. The plasmid pC4 is digested with restriction enzymes andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete IL-12/23p40 antibody is used,corresponding to HC and LC CDR regions of an IL-12/23p40 antibody of thepresent invention, respectively, according to known method steps.Isolated nucleic acid encoding a suitable human constant region (i.e.,HC and LC regions) is also used in this construct.

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 micrograms of the expression plasmid pC4 iscotransfected with 0.5 micrograms of the plasmid pSV2-neo usinglipofectin. The plasmid pSV2neo contains a dominant selectable marker,the neo gene from Tn5 encoding an enzyme that confers resistance to agroup of antibiotics including G418. The cells are seeded in alpha minusMEM supplemented with 1 microgram/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany) inalpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexateplus 1 microgram/ml G418. After about 10-14 days, single clones aretrypsinized and then seeded in 6-well petri dishes or 10 ml flasks usingdifferent concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM,800 nM). Clones growing at the highest concentrations of methotrexateare then transferred to new 6-well plates containing even higherconcentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). Thesame procedure is repeated until clones are obtained that grow at aconcentration of 100-200 mM. Expression of the desired gene product isanalyzed, for instance, by SDS-PAGE and Western blot or by reverse phaseHPLC analysis.

Example 2: Comparison of the Therapeutic Efficacy of Anti-IL-12p35 andAnti-IL-12/23p40 Antibodies in Murine Experimental AutoimmuneEncephalomyelitis (EAE)

Summary

This set of studies was performed to investigate the therapeuticefficacy of IL-12 or IL-12/IL-23 specific neutralization in a mousemodel for multiple sclerosis, experimental autoimmune encephalomyelitis(EAE). Neutralizing rat anti-mouse monoclonal antibodies (mAbs) specificfor the p35 subunit of IL-12 or the p40 subunit that is shared betweenIL-12 and IL-23 were administered either prior to disease induction,prior to disease onset, or after disease was ongoing. In all cases, onlyanti-p40 demonstrated therapeutic potential. These data suggest thatIL-23 is the predominant contributor to disease pathogenesis in thisautoimmune model.

Abbreviations

-   -   IL Interleukin    -   mAb Monoclonal antibody    -   EAE Experimental autoimmune encephalomyelitis    -   Th T helper cell    -   IFNγ Interferon gamma    -   cs Clinical score    -   MBP Myelin basic protein    -   PK Pharmacokinetics

Introduction

Biologically active IL-12 exists as a heterodimer comprised of 2covalently linked subunits of 35 (p35) and 40 (p40) kilo Daltons.Several lines of evidence have demonstrated that IL-12 can induce robustTh1 immune responses that are characterized by production of IFN□ andIL-2 from CD4⁺ T cells. Inappropriate Th1 responses, and thus IL-12expression, are believed to correlate with many immune-mediateddiseases, such as multiple sclerosis, rheumatoid arthritis, inflammatorybowel disease, insulin-dependent diabetes mellitus, and uveitis. Inanimal models, IL-12 neutralization was shown to ameliorateimmune-mediated disease. However, these studies neutralized IL-12through its p40 subunit. The recent description of IL-23 (1), aheterodimeric cytokine that shares the p40 subunit, made it important todetermine whether previous findings were due to IL-12 or IL-23 activity.Therefore, p35 and p40 specific neutralization were compared in a mousemodel of autoimmunity, experimental autoimmune encephalomyelitis (EAE).Neutralizing antibodies specific for IL-12p35 had no effect on EAEprogression. In contrast, neutralization of both IL-12 and IL-23 with ananti-p40 mAb suppressed clinical signs of EAE, whether antibody wasadministered before or after Th1 differentiation. This data suggeststhat the activity of anti-p40 treatment in EAE is based solely onneutralization of IL-23.

Methods and Materials

Mice

Female C3H/HEB/FEJ mice (Jackson Laboratories, Bar Harbor, Me.) wereused in pharmacokinetic analyses. For EAE studies, female B10.PL(H-2^(u)) mice were purchased from the Jackson Laboratories, and wereused between 6-8 weeks of age. All animals were maintained according toIACUC guidelines under approved protocols.

Antibodies

C17.8 (rat anti-mouse IL-12/IL-23p40, IgG2a), and C18.2 (rat anti mouseIL-12p35, IgG2a) hybridomas provided by Dr. Giorgio Trinchieri and theWistar Institute (Philadelphia, Pa.). Ascites was generated at HarlanBioproducts (Indianapolis, Ind.) and purified by protein G affinity.

Serum PK of Rat Anti-Mouse Antibodies

Female C3H/HEB/FEJ mice, approximately 20-25 grams, were individuallyweighed and treated with a single 5 mg/kg intraperitoneal dose of ¹²⁵Ilabeled antibody (C17.8, C18.2), with a constant dose volume/mouse of 10mL/kg. Retro-orbital bleeds were taken from anesthetized mice at 30minutes, 6 and 24 hours, 4, 7, 11 and 18 days. Blood samples wereallowed to stand at room temperature for at least 30 minutes, but nolonger than 1 hour, and were then centrifuged at approximately2,500-3,500 rpm for 10-15 minutes. Approximately 50 uL aliquots of eachserum sample were counted for ¹²⁵I using a LKB Compugamma 1282 counter(Wallac, Gaithersburg, Md.). 10 mL aliquots of the injectates were alsocounted. The average fraction of injected counts at each time point wascalculated and multiplied by the total mg of antibody injected todetermine the total mg remaining in the serum at each time point. Datais shown as the mean mg of mAb in the sera+/−s.d. with 5-10 animals ineach group.

EAE Induction and Scoring

For EAE induction, female B10.PL mice were injected subcutaneously overfour sites on the back with a total of 100 μl of CFA (containing 200 μgMycobacterium tuberculosis Jamaica strain) combined with 200 μg guineapig-MBP (Sigma). Mice also received 200 ng pertussis toxin (ListBiological, Campbell, Calif.) i.p. in 0.2 ml PBS at the time ofimmunization and 48 hours later. Mice received i.p. injections of C17.8(anti-IL-12p40) or C18.2 (anti-IL-12p35) monoclonal antibodies dilutedto 100 mg/kg (C18.2) or 20 mg/kg (C17.8) in PBS, on indicated days.Control mice received PBS or Rat IgG (Biosource) at 20 mg/kg in PBS.

Animals that demonstrated clinical signs (cs) were scored as follows:limp tail or waddling gait with tail tonicity 1, waddling gait with limptail (ataxia) 2, ataxia with partial limb paralysis 2.5, full paralysisof one limb 3, full paralysis of one limb with partial paralysis ofsecond limb 3.5, full paralysis of two limbs 4, moribund 4.5, death 5.Animals that scored a 5 were not included in the mean daily cs analysisfor the rest of the experiment. Daily cs are averaged for the group, andmean incidence, day of onset, highest acute cs, cumulative cs, cs/day,number of relapses and relapse severity ±sem are described. Meancumulative cs per group was calculated by averaging the sum of dailyclinical scores for individual animals. Cs/day was calculated bydividing the cumulative cs by the number of days the animal remained inthe study. To determine the mean day of onset, animals not developingEAE were not included in the analysis. To determine the mean highest cs,mice not developing EAE were assigned a value of “0” and included in theanalysis. Relapses were defined by a full point drop in clinical scoresustained for at least 2 observed days followed by a full point increasein clinical score sustained for at least 2 observed days.

Results and Discussion

Anti-p35 and Anti-p40 Antibodies have Identical Pharmacokinetics

To establish the clearance rates of anti-p40 and anti-p35 antibodies,normal mice were injected with a single 5 mg/kg dose of ¹²⁵I labeledantibodies and circulating levels were measured for 11 days postantibody administration. Anti-p35 and anti-p40 had overlappingpharmacokinetics, demonstrating that clearance rates are identical innormal mice (2). The expected clearance rate of each mAb isapproximately 7-10 days. Although this is a single dose PK study, thesedata support once weekly dosing for in vivo studies.

Only Anti-p40 Treatment Prior to EAE Induction is Protective

To determine the relative roles of IL-12 and IL-23 in immune-mediateddiseases, we utilized a murine model for multiple sclerosis, relapsingexperimental autoimmune encephalomyelitis (EAE). Upon EAE induction withmyelin basic protein (MBP) in adjuvant, B10.PL mice typically exhibit aninitial episode of paralysis (acute disease), then recover eitherpartially or completely and progress through multiple relapses and/orchronic EAE. It has long been assumed that EAE is dependent upon IL-12expression since IL-12 is believed to be a primary mediator of Th0 toTh1 differentiation. However, to distinguish the potential role of IL-23in EAE induction, neutralizing concentrations of anti-p40 (IL-12 andIL-23) or anti-p35 (IL-12 only) antibodies were established one dayprior to immunization for EAE (Day −1). Onset of disease can varybetween animals; therefore, treatment was repeated 7 and 14 days laterto ensure that anti-p35 and IL-p40 antibodies were present during Th1differentiation. Several in vitro neutralization studies havedemonstrated that the anti-40 mAb is 5 times more effective inneutralizing IL-12 than the anti-p35 mAb (data not shown). Therefore,the dose of anti-p35 mAb was adjusted to be 5 fold higher than anti-p40in all EAE experiments. In two separate experiments, mice treated withRat IgG isotype control antibody (20 mg/kg) or anti-p35 (100 mg/kg) didnot demonstrate protection from disease. It is important to note thatperipheral administration of a non-specific control antibody (Rat IgG)did not alter the clinical course of disease when compared tonon-treated mice with EAE. In both studies, mice treated with anti-p40mAb (20 mg/kg) exhibited nearly complete inhibition of EAE clinicalsigns. Remarkably, suppression of disease extended beyond the expectedrate of antibody clearance through 70 days post EAE induction. In eachexperiment, only one animal treated with anti-p40 exhibited twoconsecutive days of EAE clinical signs, and each demonstrated a lateonset and significantly lower acute clinical scores, cumulative clinicalscores, and no relapses in disease (Table 1). These results demonstratethat neutralization of IL-12 and IL-23 through the shared p40 subunitprovided nearly complete protection from EAE. In contrast, specificneutralization of IL-12 only via anti-p35 was ineffective. These datastrongly suggest that EAE is not mediated by IL-12.

Only Anti-p40 Treatment Just Prior to Disease Onset is Protective

Although prophylactic treatment completely protected mice from EAE, itremained to be determined if IL-12 specific neutralization would beprotective once the Th1 population was established in vivo. Therefore,in a separate set of experiments, mice were treated with either acontrol antibody (Rat IgG), anti-p35, or anti-p40 monoclonal antibodiesten days after EAE induction, but prior to disease onset. Since typicalimmune responses occur within 7 days, this time point should reflect theeffects of anti-IL-12 or anti-IL-23 mAbs on differentiated Th1 cells.EAE onset can vary between animals, therefore treatment was repeated 7and 14 days later to ensure that anti-p35 and anti-p40 antibodies werepresent during the onset of disease. In two separate experiments, micetreated with isotype control antibody (20 mg/kg) or anti-p35 (100 mg/kg)were not protected from disease, when compared to untreated EAE mice.However, mice treated with anti-p40 mAb (20 mg/kg) were significantlyprotected from EAE. As shown in the previously described studies,disease suppression was observed well beyond the time required forclearance of peripherally administered antibody through day 70 post EAEinduction. Considering that antibody was not administered until afterTh1 differentiation (day 10), it was not surprising that diseaseincidence, day of onset, and the highest clinical score during acute EAEwere not different in any group (Table 2). However, in both experiments,mice receiving anti-p40 exhibited significantly lower cumulativeclinical scores, clinical scores per day, and relapse severity.

Only Anti-p40 Treatment During Established EAE is Protective

The most difficult, but clinically relevant, hurdle for any therapy isto suppress established disease. Therefore another set of experimentswas performed in which mice were immunized for EAE, then divided intotreatment groups once disease was ongoing. Approximately 30 days postEAE induction, mice had progressed through the acute phase of disease.At this time, animals were divided into groups with comparablecumulative and daily clinical scores. Treatment was repeated 7 and 14days later to ensure that antibodies were available in neutralizingconcentrations during the transition from acute to chronic orremitting-relapsing disease. Only anti-p40 treatment (20 mg/kg)ameliorated disease when compared to either isotype control antibody (20mg/kg) or anti-p35 (100 mg/kg) treated animals. Disease suppression wasobserved through day 80 post EAE induction. In both experiments,analysis from the first day of treatment through day 80 demonstratedthat mice receiving anti-p40 exhibited lower cumulative clinical scores,clinical scores per day, and the least highest clinical score posttreatment. These data suggest that not only is IL-23 likely to mediateTh1 differentiation (Table 1) and EAE induction (Table 2), but IL-23also contributes to the effector phase of chronic immune-mediated (e.g.,autoimmune) responses (Table 3). Therefore, anti-p40 treatment can offertherapy at any time in the progression of immune-mediated diseases.

Conclusions

The understanding of the role of IL-12 in immune function has been basedon studies of the p40 subunit of IL-12. Therefore, a side-by-sidecomparison of neutralization of the IL-12 specific p35 subunit versusthe p40 subunit shared between IL-12 and IL-23 was conducted in ananimal model of autoimmune disease. Neutralization via anti-p40significantly inhibited EAE when mAb was administered at any time point.However, IL-12 specific neutralization was completely ineffective.Therefore, our data shows that IL-12 does not contribute to thisautoimmune model and that IL-23 is expected be the more prominentmediator of autoimmune T cell responses.

Example 3: p40 Neutralizing Epitope

Summary

The epitope for a neutralizing antibody (IL-12/23p40 mAb) against humanp40 subunit of IL-12 and IL-23 was determined based on the crystalstructure of the Fab/IL-12 complex. The epitope is located on the D1domain (residues 1-88) of the p40 subunit of human IL-12. This region isdistant from the p40/p35 interface and is expected to be also availableon the p40 subunit of IL-23. The residues involved in antigen-antibodybinding are discontinuous (Table 4) and comprise a unique conformationalepitope. Antibodies against this epitope or portions of it andneighboring regions will lead to blockage of IL-12 and IL-23 functionsmediated through this part of p40 subunit.

Introduction

A fully human monoclonal antibody (p40 mAb) directed against humanIL-12/23p40 has been shown to be a potent neutralizer of IL-12 and IL-23function. It has been shown that the p40 mAb binds to the p40 subunitand blocks the binding of both cytokines to their receptors. Because thep40 subunit is shared between IL-12 and IL-23, the detailed interactionsbetween IL-12 and the p40 mAb define an important common neutralizingepitope, which may in turn shed some light on the cytokine-receptorinteractions.

Epitope Determination Based on p40 Fab/IL-12 Crystal Structure

The IL-12/IL-23p40 mAb was produced from a mammalian cell line inculture and purified by protein A column. The IL-12/IL-23p40 mAb (70 mg)was digested with papain (0.25 units of papain per milligram of IgG) inactivation buffer (0.03M sodium phosphate, 0.15M NaCl, 0.01M EDTA,0.0072M L-cysteine, pH 7.0) at 37° C. for 2 hours. Digestion wasmonitored by Surface-Enhanced Laser desorption ionization (SELDI) massspectrometry. Iodoacetamide (0.5M) was used to stop the digestion. Fcwas removed by immobilized protein G. The p40 Fab was further purifiedby gel filtration on a Superdex 200 16/60 column. A total of 44 mgpurified p40 Fab was obtained and its purity analyzed by SDS-PAGE.

Recombinant human IL-12 was produced in culture from a stablytransfected cell line over-expressing the p40 and p35 subunits andpurified with a p40 mAb affinity column. The protein fractions werecollected and dialyzed into 10 mM Tris, 100 mM NaCl, pH 7.4 andconcentrated to 2.5 mg/ml. IL-12 was deglycosylated by incubating withseveral combinations of deglycosylation enzymes (PNGase F, Sialadase,Endo-O-glycosydase, α-, β-galactosidase, α-mannosidase, fucosidase[about 5 mU-10U/100 ug protein]) for 3 days at 37° C. under argon.

Deglycosylated IL-12 was mixed with an excess of the p40 Fab. TheIL-12/p40 Fab complex was purified by size exclusion chromatography in10 mM Tris, 50 mM NaCl, pH 7.4. The isolated complex was concentrated toapproximately 4 mg/ml. The IL-12/p40 Fab complex was crystallized usingthe sitting drop vapor diffusion method by combining the above proteincomplex solution in 1:1 volume ratio with a reservoir solution of 50 mMTris, pH 7.0, 16% PEG 3350. Cubic, pyramidal or rod shaped crystals oftypically 50-150 μm in size appear within two weeks at 16° C.

The crystals were harvested, and soaked in the mother liquor plus 30%ethylene glycol and flash frozen in liquid nitrogen for X-ray datacollection. The best data set was collected (360 degrees, 0.5degree/frame, 10 second exposure per frame) to a diffraction limit of2.8 Å at Advanced Photon Source (APS), Argonne National Laboratory,(Axas-ComCat). The diffraction data were processed with Denzo andScalePack. (Otwinowski & Minor, Methods Enzymol. 276: 307-326) The spacegroup for this crystal form is P212121 with cell dimensions of a=116.8Å, b=55.77 Å, c=182.96 Å, and α=β=γ=90°. There are 27,141 independentreflections and the data was ˜90% complete at 3.0 Å (I/sigma=5.2,R_(sym)=9%.).

The crystal structure was solved by molecular replacement as implementedin CNX (Accelrys, Calif.). The search models were the published IL-12structure (PDB code 1F45) for IL-12 and a homology model for p40 Fabbased upon a Fab crystal structure (PDB code 1VGE). The molecular modelsfor IL-12 and the p40 Fab were visually inspected and manually adjustedusing XtalView. The structure refinement was carried out with the CNX.The molecular models were validated with InsightII (Accelrys, Calif.).The molecular structure of the bound complex of IL-12/p40 Fab is shownin FIG. 1 in a ribbon representation.

The p40 mAb binding site (p40 epitope) on the molecular surface insurface and ribbons representations is shown in FIG. 2. The bindingepitope residues are defined as any surface exposed p40 residues(relative solvent accessibility of 0.1 or greater) with any atoms within4 Å of any atoms of the antibody, according to a generally acceptedconvention. The surface accessibilities were calculated by ICM (Molsoft,Calif.) using the default parameters. Residues comprising the p40 mAbbinding epitope on IL-12 p40 are listed in Table 4, along with theirsurface exposed areas (sf) and relative accessibilities (sf ratio). Thedefinition of exposed surface area of an amino acid residue within thecontext of protein has been well accepted in the field. In particular, awater molecule with a radius of 1.4A is rolled along the surface of aprotein and the amount of area obtained by this calculation for aspecific residue is assigned to the exposed surface area of thatresidue. The total surface area for an amino acid in fully extendedconformation is also available. The surface accessibility (sf ratio) isthen the ratio of the exposed surface area over the standard surfacearea. These two values together give us ideas whether a residue isexposed on the protein surface.

It is clear from Table 4 and FIG. 2 (right panel) that the binding siteon p40 is discontinuous and constitutes a large number of surfaceexposed residues that are distributed over an irregular surface. Theantibody-antigen interaction buries a total of 1758 Å² of accessiblesurface on IL-12 and p40 mAb. The interactions appear to be dominated bythree salt bridges: R59(H)-E59(p40), R98(H)-E45(p40) andR99(H)-D62(p40). Also, there are contributions to the antibody-antigeninteraction that are due to hydrophobic or Van der Waals forces.

The residues on the IL-12p40 antibody involved in IL-12 bindingidentified based upon the co-crystal structure herein are shown in Table5 below. All surface exposed residues of the anti-p40 antibody with anyatoms within 4A of the p40 subunit are considered part of these bindingresidues. Conservative changes to any one or more of these residues mayproduce mutant antibodies that are of similar potency. Examples of suchconservative substitutions include, without limitation, R59K, R98K andR99K in VH (e.g., SEQ ID NO:7) and D1E in VL (e.g., SEQ ID NO:8).

Also, saturation mutagenesis at each position shown in Table 5 can beperformed (e.g., changing the wild type (WT) amino acid sequence to anyother amino acid, possibly with the exception of Cysteine) to identifymutations causing the resulting antibody to have increased, decreased orsubstantially similar activity (e.g., binding). Binding of the resultingantibody to the p40 subunit can be tested pursuant to any suitablebinding assay. Saturation mutagenesis can be used, for example, tocreate a more or less potent antibody or an equally potent antibodyhaving different properties, i.e., properties other than potencyresulting from a change in the variable region sequence (e.g., size orother structural changes to the variable region).

The saturation mutagenesis can also be performed using more than onesubstitution, for example, two, three, or more positions for eachexperiment. This can be done as individual clones or libraries followedby selection or screening in an appropriate format, such as phagedisplay. Furthermore, individual or combination mutations at thepositions in Table 5 that are desirable to regulate activity can becombined to create additional combination mutants of similar or betterpotency.

Advantage

The p40 mAb binding site is distant from the p40/p35 association site(FIG. 1). The p19 subunit of IL-23 is evolutionarily related to p35 ofIL-12 with significant sequence homology. It is likely that p19associates with p40 in a fashion similar to p35. Therefore, the p40 mAbbinding region is also distant from the p40/p19 interaction in IL-23.The epitope identified in the present invention is equally available inboth IL-12 and IL-23; accordingly, it is not surprising that p40 mAb canactively block the functions of both cytokines.

IL-12 (p40/p35) and IL-23 (p40/p19) interact with their respectivereceptors (IL-12Rβ1/β2 and IL-12Rβ1/IL-23R) in a similar fashion. Theyinduce similar signaling cascades. The details of the cytokine-receptorinteractions, however, are not clearly defined at the molecular level.The newly defined p40 mAb epitope could represent a biologicallyimportant site for the interactions between the IL-12 family ofcytokines with their shared receptor, IL-12Rβ1. Therefore, the epitopeis an important target for the therapeutic intervention using monoclonalantibodies, peptides, recombinant proteins, small molecules and othernatural or synthetic agents.

Example 4: Epitope Mapping of p40 mAb on IL-12p40 Using MutationalAnalysis

Summary

ELISA binding of IL-12p40 mutants with IL-12/IL-23p40 mAb was carriedout to verify the binding epitope. Based on the crystal structure of p40Fab/IL-12 complex, 7 single mutants and 2 double mutants were generated.The mutated residues are located in the p40 Fab contact region in thedomain I (D1) of p40 subunit. The relative binding affinity showed thatthree negatively-charged residues, E45, E59 and D62, contributesignificantly to binding interactions with p40 mAb. The other residues,M23, L40 and S43, have less but appreciable contribution. Thismutational analysis confirms that p40 mAb recognizes domain I of p40subunit and residues E45, E59, D62, M23, L40, and S43 are part of thebinding epitope.

Materials and methods

Seven human p40 single mutants, M23T, L40T, S43R, E45A, E45R, E59R, andD62R, and two double mutants, S43R/E45A and S43R/E45R, are used in thestudies. The wide-type human p40, WT hu-p40, was used as the control.The p40 mutants were transiently expressed in HEK293E cells. Thesupernatants were used for binding assays.

In brief, MSD high bind plates (Meso Scale Discovery, MD) were coatedwith 5 μl of a capture monoclonal antibody (5 μg/ml) at room temperaturefor 1 hour. The capture monoclonal antibody recognizes p40 subunit butdoes not compete with p40 mAb. One-hundred and fifty (150) μl of 5% MSDBlocker A buffer was added to each well and incubated for 1 hr at roomtemperature. Plates were washed three times with 0.1 M HEPES buffer (pH7.4). These protein-charged ELISA plates were incubated with 25 μl ofdifferent transiently expressed p40 mutant supernatants (1:10 dilutionwith 0.1 M HEPES buffer, pH 7.4) for 1 hr at room temperature. Theplates were washed three times with 0.1 M HEPES buffer (pH 7.4).Twenty-five (25) μ1 of different concentrations of MSD Sulfo-TAG labeledp40 mAb, ranging from 0 to 20 μg/ml, were dispensed to micro-wells.After incubation for 2 hrs shaking at room temperature, plates werewashed 3 times with 0.1 M HEPES buffer (pH 7.4). One hundred and fifty(150) μ1 of diluted MSD Read Buffer T was dispensed into each well andthe plates were analyzed with a SECTOR imager (MSD).

Results

As shown in FIG. 3, E45R, E59R, and D62R each reduced the mutein p40binding affinity to p40 mAb significantly, compared to the wild typep40. E45A had significant but less dramatic effect on the bindingcompared to E45R. M23T, L40T and S43R each had moderate effect uponbinding.

The relative binding affinity of IL-12/IL-23p40 mAb to different mutantproteins was further analyzed by a capture assay. As shown in FIG. 4,E45R, E59R and D62R each nearly completely abolished antigen binding.The other mutations, M23T, L40T and S43R, in the interface, each reducedbinding by approximately 40%. E45A reduced binding by about 70%.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

TABLE 1 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day -1 (prior to Th1 differentiation). Group IncidenceMortality Day of onset Highest acute cs^(a) Cumul cs^(b) Cs/day No. ofrelapses Relapse severity P-2001-060 Rat IgG 13/13 4/13 30.5 ± 3.2 3.6 ±0.3 71.4 ± 14.1 1.2 ± 0.2 1.3 ± 0.2 3.6 ± 0.2 Anti-p35 11/13 8/13 29.6 ±3.4 3.5 ± 0.5 45.5 ± 11.5 0.8 ± 0.2 1.2 ± 0.1 4.0 ± 0.3 Anti-p40  1/130/13 40.0 0.1 1.2 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 P-2001-079 Notreatment 6/7 0/7  24.7 ± 2.7 3.2 ± 0.6 110.4 ± 20.4  1.7 ± 0.3 1.0 ±0.4 3.8 ± 0.1 Rat IgG 9/9 2/9  29.1 ± 2.9 3.8 ± 0.2 90.6 ± 10.1 1.5 ±0.1 0.3 ± 0.2 4.7 ± 0.3 Anti-p35 10/10 1/10 30.0 ± 2.6 3.9 ± 0.2 94.9 ±17.8 1.4 + 0.2 0.7 ± 0.3 3.9 ± 0.2 Anti-p40  1/10 0/10 61.0 0.3 1.6 ±1.1 0.0 ± 0.0 0.0 + 0.0 0.0 + 0.0 ^(a)clinical score (cs) ^(b)cumulativecs Mice were treated as described and clinical scores were analyzed fromday 0 through 70 days post EAE induction. Data is shown as the mean pergroup ± s.e.m.

TABLE 2 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day 10 (after Th1 differentiation). Group IncidenceMortality Day of onset Highest acute cs^(a) Cumul cs^(b) Cs/day #relapses Relapse severity P-2001-037 No treatment 7/8 0/8  30.6 ± 2.73.2 ± 0.5 51.5 ± 14.4 0.8 ± 0.2 0.3 ± 0.2 3.3 ± 0.8 Rat IgG  9/10 0/1025.9 ± 2.7 2.7 ± 0.5 74.7 ± 15.8 1.2 ± 0.2 0.6 ± 0.2 3.7 ± 0.4 Anti-p35 9/10 0/10 25.8 ± 2.6 2.5 ± 0.4 58.8 ± 15.6 1.0 ± 0.2 0.7 ± 0.3 3.2 ±0.3 Anti-p40 6/7 0/7  34.7 ± 6.3 1.6 ± 0.5 14.9 ± 7.5  0.2 ± 0.1 0.3 ±0.2 1.5 ± 0.5 P-2001-053 No treatment 8/9 2/9  15.8 ± 2.2 2.1 ± 0.6 56.4± 19.1 0.9 ± 0.3 0.6 ± 0.3 3.3 ± 0.5 Rat IgG  9/10 4/10 20.0 ± 2.5 3.8 ±0.5 70.1 ± 17.7 1.3 ± 0.2 0.3 ± 0.2 4.2 ± 0.4 Anti-p35 10/10 1/10 16.5 ±1.1 3.2 ± 0.3 93.8 ± 15.7 1.4 ± 0.2 0.8 ± 0.2 3.2 ± 0.3 Anti-p40 10/102/10 13.6 ± 1.1 2.7 ± 0.5 23.2 ± 7.9  0.4 ± 0.1 0.4 ± 0.3 2.0 ± 0.4^(a)clinical score (cs) ^(b)cumulative cs Mice were treated on days 10,17, and 24 and clinical scores were analyzed from day 0 through 70 dayspost EAE induction. Data is shown as the mean per group ± s.e.m.

TABLE 3 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day 30 (during established EAE). From first treatmentthrough 80 days post EAE induction Group Pre-Tx^(a) Mortality Cumulcs^(b) Cs/day Highest cs Lowest cs # relapses Relapse severity P-2002-01No treatment 2.7 ± 0.6 1/5 132.9 ± 29.3 3.3 ± 0.3 4.1 ± 0.2 2.4 ± 0.50.6 ± 0.4 3.7 ± 0.0 Anti-p35 2.3 ± 0.7 1/5 135.9 ± 16.5 2.7 ± 0.3 3.8 ±0.4 1.8 ± 0.3 2.0 ± 0.4 3.7 ± 0.3 Anti-p40 2.0 ± 0.2 1/6  75.6 ± 16.11.9 ± 0.3 2.8 ± 0.5 1.0 ± 0.4 0.7 ± 0.3 2.5 ± 1.0 P-2002-093 Rat IgG 1.7± 0.8 1/5  87.7 ± 16.4 2.1 ± 0.2 3.7 ± 0.4 1.2 ± 0.5 1.5 ± 0.5 3.8 ± 1.0Anti-p35 1.9 ± 0.7 1/5 98.2 ± 9.7 2.2 ± 0.1 3.7 ± 0.4 1.4 ± 0.4 1.5 ±0.3 3.3 ± 0.2 Anti-p40 2.4 ± 0.8 0/5  71.7 ± 21.6 1.5 ± 0.4 2.9 ± 0.60.8 ± 0.5 1.3 ± 0.3 2.7 ± 0.6 ^(a)mean clinical score per group on thefirst day of treatment (Tx) ^(b)clinical score (cs)

TABLE 4 Residues contributing to the p40 mAb binding epitope onIL-12/p40 Number Residue sf Sf Ratio 15 Trp 94.2 0.36 17 Pro 69.3 0.4618 Asp 132.9 0.86 19 Ala 27.7 0.22 20 Pro 131.5 0.88 21 Gly 18.7 0.21 23Met 142.4 0.66 40 Leu 32.7 0.16 41 Asp 69 0.45 42 Gln 145.8 0.77 43 Ser58.2 0.46 45 Glu 139.2 0.74 46 Val 95.8 0.57 47 Leu 95 0.48 54 Thr 77.20.51 55 Ile 32.7 0.17 56 Gln 111 0.58 58 Lys 69.2 0.32 59 Glu 83.2 0.4460 Phe 113.3 0.51 61 Gly 68.2 0.77 62 Asp 41.3 0.27

TABLE 5 Residues on p40 mAb involved in binding to IL-12/p40 epitopeHeavy chain residues 1. Light chain residues S28 D1 T31 S30 Y32 W32 W33Y49 D57 S56 R59 N92 R98 I93 R99 Y94 R100 P101 G102 Q103

Example 5: Results of Clinical Trials Using Increased Dosing Intervals

Ustekinumab was studied in a Phase 3b, randomized, double-blind, activetreatment-controlled, multicenter study with a 4-week screening period,an open-label run-in period from Week 0 to Week 28, a double-blindtreatment period from Weeks 28 to Week 104, a post-treatment periodthrough 116, and a safety follow-up via contact by telephone or anonsite visit at Week 124 (see FIG. 5: Study Schema). The study wastitled “Phase 3b, Randomized, Double-blind, Active-controlled,Multicenter Study to Evaluate a “Subject-tailored” Maintenance DosingApproach in Subjects with Moderate-to-Severe Plaque Psoriasis(PSTELLAR)” under Protocol Number CNTO1275PS03009. In FIG. 5,NR=nonresponder at a given timepoint; R=randomization of responders[defined as a PGA of <2] at Week 28 (subjects who are nonresponders[defined as a PGA of ≥2] at Week 28 will be discontinued from receivingfurther study agent injections, will be followed for safety for at least20 weeks after their last study agent injection, and then will bewithdrawn from the study); UST=ustekinumab; Wk(s)=Weeks.

All subjects enrolled were exposed to ustekinumab. At each injectionvisit prior to Week 28, subjects with baseline weight of ≤100 kgreceived one injection (ustekinumab 45 mg) and those with baselineweight >100 kg received 2 injections (ustekinumab 45 mg+ustekinumab 45mg). At each injection visit from Week 28 to Week 104, subjects withbaseline weight of ≤100 kg received one injection (ustekinumab 45 mg orplacebo) and those with baseline weight >100 kg received 2 injections(ustekinumab 45 mg+ustekinumab 45 mg or placebo+placebo) depending ontreatment group assignment and injection visit (see Table 7: Schedulefor Study Agent Administration). The weight-based dose of study agentdetermined at baseline was utilized throughout the study. At Week 28,responders, defined as those subjects with a PGA of cleared (0) orminimal (1) were randomized with a 4 to 1 ratio into 2 groups, whilenon-responders (PGA score >2) discontinued from the study altogether.

-   -   Group 1 (n=76): Beginning at Week 28, subjects received        fixed-interval maintenance dosing of ustekinumab q12w through        the end of the study (i.e., Weeks 28, 40, 52, 64, 76, 88, and        100).    -   Group 2 (n=302): Beginning at Week 28, subjects received a        tailored interval maintenance dosing. Using placebo injections,        the period between study drug injections were extended in order        to define their individually-tailored maintenance dosing        interval. The study drug dosing interval for each subject was        determined by assessing for maintenance of response without        receiving a study drug injection at increments of 4 weeks until        loss of PGA response was observed. At that point, subjects then        assumed a study drug dosing interval defined by the longest        interval for which a PGA response had been maintained (i.e., 4        weeks shorter than the time point at which loss of response was        noted) or a maximal dosing interval of 24 weeks if response was        maintained through the 24 weeks. Therefore, four different dose        intervals were explored among subjects in Group 2, with        respective treatment groups designated as Groups 2a, 2b, 2c and        2d:        -   a. With first loss of response (PGA score of ≥2) 16 weeks            after the Week 16 injection (Week 32 visit), subjects would            then receive ustekinumab q12w starting at Week 32 through            Week 104 dosing visits (i.e., Weeks 32, 44, 56, 68, 80, 92,            and 104).        -   b. With first loss of response (PGA score of ≥2) 20 weeks            after the Week 16 injection (Week 36 visit), subjects would            then receive ustekinumab q16w starting at Week 36 through            Week 104 dosing visits (i.e., Weeks 36, 52, 68, 84, and            100).        -   c. With first loss of response (PGA score of ≥2) 24 weeks            after the Week 16 injection (Week 40 visit), subjects would            then receive ustekinumab q20w starting at Week 40 through            Week 104 dosing visits (i.e., Weeks 40, 60, 80, and 100).        -   d. With no loss of response (PGA score of <2) 24 weeks after            the Week 16 injection (Week 40 visit), subjects would then            receive ustekinumab q24w starting at Week 40 through Week            104 dosing visits (i.e., Weeks 40, 64, and 88).

During the double-blind treatment period, subjects in Groups 1 and 2received placebo injections as necessary to maintain the blind withregard to dosing interval. Subjects in Group 2 were to receive at least3 cycles of subject-tailored Interval maintenance dosing during therandomized study period (see Table 7: Schedule for StudyAdministration).

-   -   Randomization: At Week 28, subjects with static PGA score of        cleared (0) or minimal (1) were randomized in a 1:4 ratio to        either Group 1 (the approved q12w maintenance regimen) or Group        2 (“subject-tailored” interval maintenance regimen) by using the        randomly permuted blocks. The randomization was stratified by        subject body weight at baseline [≤100 kg vs >100 kg] and PGA        score [0 or 1] at Week 28.    -   Treatment duration/Trial duration: Subjects were dosed with        study agent from Week 0 through Week 104. Subjects were then        followed for at least an additional 20 weeks, with a final study        visit at Week 124.    -   Primary analysis set for efficacy: The primary efficacy analysis        was based on all subjects who were randomized to one of two        treatment regimens (Group 1 or Group 2) at Week 28.    -   Primary efficacy variable/Primary time point: The primary        endpoint is the number of visits at which subjects achieved a        static PGA score of cleared (0) or minimal (1) between Week 88        and Week 112 (evaluation interval) for subjects randomized at        Week 28. Assessment visits were conducted every 4 weeks during        the evaluation interval.    -   Major secondary efficacy variables:        -   The proportion of subjects with a static PGA score of            cleared (0) or minimal (1) and its 95% confidence interval            by visit from Week 28 to Week 112 for subjects randomized at            Week 28.        -   The number of visits for which subjects achieved a PASI 75            response between Week 88 and Week 112 for subjects            randomized at Week 28.        -   The proportion of subjects with a PASI 75 response and its            95% confidence interval by visit from Week 28 to Week 112            for subjects randomized at Week 28.    -   Expected effect size and planned sample size: Based on data from        PHOENIX 1 and PHOENIX 2, it was anticipated that approximately        35% of 500 enrolled subjects would be ineligible for        randomization at Week 28 because of study discontinuation prior        to Week 28 or the lack of a PGA score of cleared (0) or        minimal (1) response at Week 28. The anticipated 325 subjects        eligible for randomization at Week 28 in a 1:4 ratio would        provide approximately 65 subjects in Group 1 (q12w maintenance        dosing) and approximately 260 subjects in Group 2        (subject-tailored maintenance dosing).

The study was designed to estimate the response rates for 2 ustekinumabmaintenance groups in greater than 300 subjects. A total ofapproximately 325 randomized subjects would provide a 95% confidenceinterval of 64.9% to 85.9% around a response of 75% in Group 1 (n=65;q12w maintenance dosing) and a 95% confidence interval of 54.0% to 66.0%around a response of 60% in Group 2 (n=260; subject-tailored maintenancedosing) during the evaluation period between Weeks 88 and 112.

Primary Objective:

The primary objective of this study was to assess the effect ofextending maintenance dosing intervals beyond every 12 weeks (q12w) onthe clinical efficacy of ustekinumab.

Topline Results Summary

CNTO1275PS03009 is a Phase 3b, randomized, double-blind, activetreatment-controlled, multicenter trial in adult subjects ≥18 to ≤80years of age with moderate to severe plaque psoriasis, defined by PGA ≥3and BSA involvement of at least 10%, who were candidates forphototherapy or systemic treatment of psoriasis.

The study began with its first subject screened on Mar. 8, 2012. A totalof 611 subjects were screened, of which 478 subjects were enrolled intothe study, and 378 subjects were randomized at Week 28 into the twotreatment groups, one receiving the fixed q12 week interval maintenanceregimen (Group 1) and the other receiving a subject-tailored maintenancedose interval regimen (Group 2). The study was conducted at 42 sites inUSA. The final database lock includes data through Week 124 for allenrolled subjects. The first subject was dosed on Mar. 22, 2012. Thefirst subject was randomized on Oct. 4, 2012 and the last subject wasrandomized on Sep. 16, 2013.

A total of 611 subjects were screened and 478 subjects were ultimatelyenrolled into the study. A total of 378 responders (PGA 0/1) wererandomized at Week 28 (80% randomization rate). This randomization ratewas higher than expected, which could be, in part, due to the subjectivenature of the PGA scoring at Week 28. This final database lock includeddata through Week 124 for all enrolled subjects.

Distribution of Subjects by Treatment Groups and Subject Evaluability

A total of 478 subjects were enrolled and treated with ustekinumabthrough the open-label period, Week 0-28 run-in period, using theapproved US weight-based dosing guidelines (ustekinumab 45 mg, n=308;ustekinumab 90 mg, n=170). A total of 378 subjects were randomized into2 groups at Week 28: a fixed-interval every 12 week maintenance dosinggroup (Group 1) and a subject-tailored interval maintenance dosing group(Group 2). Using a 1:4 randomization ratio, a total of 76 subjects wererandomized into Group 1 and 302 subjects were randomized into Group 2.The distribution of patients in Group 2 receiving maintenance dosing foreach of the 4 potential dosing intervals was: q12w, n=84; q16w, n=61;q20w, n=51; and q24w, n=84.Subject demographics

Among overall enrolled subjects, the majority were white (84.9%), 63.0%were male and the median age was 46 years. Among all randomizedsubjects, most were white (76.7% for Group 1 and 89.4% for Group 2) andmale (57.9% for Group 1 and 63.6% for Group 2). The median age was 42years for Group 1 and 46 years for Group 2.

Subject Disease Characteristics

Among overall enrolled subjects, at baseline the median duration ofpsoriasis was 13.3 years, the median percent of body surface area (BSA)involved was 19.0%, the median PASI score was 16.0. A total of 35.8% ofsubjects presented with a PGA ≥4 consistent with severe disease;therefore, the majority of the subjects had moderate disease as definedby baseline PGA score of 3. Baseline disease characteristics ofrandomized subjects in Groups 1 and 2 were generally comparable, andwere similar to those of the overall enrolled population. Howeversubjects in Group 1, on average, had longer duration of disease (17.5years) and higher baseline BSA of involved skin (21.0%) compared tosubjects in Group 2 (11.8 years and 17.0% BSA, respectively).

Subject Psoriasis Medication History

Among overall enrolled subjects, 32.4% previously received phototherapy,35.4% previously received conventional systemic therapy (including PUVA,methotrexate, acitretin, cyclosporine, mycophenolate mofetil) and 31.0%previously received biologic therapy. Generally, similar proportionswere observed among subjects randomized to Groups 1 and 2. Among overallenrolled subjects, 69.0% were naïve to prior biologic use. Prior use ofconventional systemic agents was slightly higher among subjects in Group1 39.5% compared to Group 2 (31.1%). A higher proportion of subjects inGroup 2 (74.5%) were naïve to prior biologic use compared to Group 1(64.5%).

Discontinuation of Study Agent

During the open-label period (Week 0 through Week 28), 20.9% (100/478)of enrolled subjects discontinued use of study agent. Generally, similarproportions of subjects discontinued study agent in the 45 mg group(19.8%) and in the 90 mg group (22.9%). The most common reason fordiscontinuation was failure to achieve a static PGA score of cleared (0)or minimal (1) at Week 28 [12.1% (58/478)] to be eligible advance to therandomized portion of the study. During the randomized dosing period(Week 28 through Week 104), 22.2% (84/378) of randomized subjectsdiscontinued study agent. The most common reason for discontinuation ofstudy agent was loss to follow-up (7.9%) among Group 1 subjects andadverse event (5.0%) or withdrawal of consent (5.0%) among Group 2subjects.

During the randomized treatment period (Week 28 to Week 104), 22.2%(84/378) of randomized subjects discontinued the study agent. Rates ofdiscontinuation were similar between Groups 1 and 2 (Table 8). The mostcommon reason for discontinuation of study agent among subjects in Group1 was loss to follow-up (7.9%), and adverse event (5.0%) or withdrawalof consent (5.0%) among subjects in Group 2.

Efficacy Findings:

Primary Endpoint—

-   -   Group 2 had fewer visits (mean difference of −0.46) at which        subjects had a PGA score of cleared (0) or minimal (1) (PGA 0/1)        during the Week 88 and Week 112 evaluation interval compared to        Group 1 (on average, Group 1 had 4.5 visits with PGA 0/1, and        Group 2 had 4.1 visits with PGA 0/1).        -   Of note, during this interval Group 1 (55.3%) showed a            higher proportion of subjects with a PGA score of 0/1 at all            7 visits during the evaluation interval compared to Group 2            (38.1%).            Secondary Endpoints—    -   Group 2 had fewer visits (mean difference of −0.32) at which        subjects had a PASI 75 between Week 88 and Week 112 compared to        Group 1 (on average, Group 1 had 5.8 visits with PASI 75, and        Group 2 had 5.4 visits with PASI 75). The number of visits at        which subjects had a PASI 75 response during the evaluation        interval was similar for Groups 1 (69.7%) and Group 2 (66.9%).    -   Responses over time after randomization:        -   PGA 0/1 response rates decreased for both Groups 1 and 2            during the dose interval determination period (Week 28 to            Week 40). As expected, a greater decrease was observed for            Group 2 compared to Group 1, as defining subject-tailored            dosing intervals was based on worsening of disease for Group            2 subjects. After Week 40, the PGA response rates for both            groups were generally maintained through Week 112. Response            rates at most visits were slightly higher for Group 1            compared to Group 2.        -   In general, patterns for PASI 75 responses reflected those            for PGA responses from Week 28 through Week 112.        -   The proportions of subjects achieving PGA of cleared (0),            PASI 90 or PASI 100 responses through Week 112 were            generally higher for Group 1 compared to Group 2. The            magnitude of the difference between the Groups 1 and 2 was            greater for these endpoints compared to PGA 0/1 and PASI 75            endpoints.            Safety Findings:            For the randomized study population of 378 subjects, from            Week 28 through Week 124:    -   The proportion of subjects experiencing 1 or more AEs was        comparable between the two treatment groups (72.4%% in Group 1        and 72.8% in Group 2).    -   AEs were most frequently reported for the infections and        infestations system organ class (SOC) for both Group 1 (46.1%)        and Group 2 (48.7%; 48.1% for Groups 1 and 2 combined); the most        common AEs in this SOC were URTI (27.6% in Group 1 and 19.5% in        Group 2) and nasopharyngitis (9.2% in Group 1 and 13.2% in Group        2).    -   The proportions of subjects with one or more serious adverse        events (SAEs) were 9.2% for Group 1 and 7.0% for Group 2.    -   The proportions of subjects who discontinued study agent due to        one or more AEs (DCAE) were 6.6% for Group 1, and 5.6% for Group        2.    -   The proportions of subjects with one or more infections were        comparable between the two groups (48.7% in Group 1 and 45.7% in        Group 2). The most common infections in the combined group were        URTI (27.6% in Group 1 and 19.5% in Group 2) and nasopharyngitis        (9.2% in Group 1 and 13.2% in Group 2).        -   No serious infections were reported for subjects in Group 1;            serious infections were reported for 3 subjects Group 2.    -   There were two investigator-reported major adverse        cardiovascular events (MACE): 1 myocardial infarction (in a        subject receiving 90 mg in Group 2 at a dosing interval of q24        weeks) and 1 stroke (in a subject receiving 45 mg in Group 2 at        a dosing interval of q24 weeks).    -   Of the 378 randomized subjects there were 10 reported with at        least one malignancy: 6 subjects with non-melanoma skin cancers        (NMSC) and another 4 subjects with other malignancies.    -   No injection site reactions (ISRs) were reported among subjects        in Group 1. In Group 2, 2.0% of placebo injections and 0.4% of        45 mg injections were associated with ISRs; all were of mild        intensity.    -   Markedly abnormal changes in blood hematology and chemistry        laboratory values were uncommon.        For the overall study population of 478 subjects enrolled at        Week 0, through Week 124:    -   There were two deaths reported during the study. 1 death due to        natural causes, and the other due to acute myeloid leukemia        (AML). Neither death was reported during the open-label period        (Week 0 through Week 28).    -   Through Week 124, there were 39 subjects (8.2% of the overall        enrolled population) who experienced one or more SAEs.        -   Serious infection was reported for 7 subjects (1.5%).        -   No opportunistic infections or cases of active TB were            reported.    -   Through Week 124, 7.3% of subjects among overall enrolled        subjects discontinued study agent due to 1 or more AEs.    -   No possible anaphylactic or possible serum sickness-like        reactions associated with ustekinumab were reported during the        study.    -   A total of 12 enrolled subjects (2.5%) reported one or more        malignancies (including NMSC and other malignancies): 5 subjects        with BCC, 4 subjects with cutaneous SCC, 6 subjects with other        malignancies    -   There were 3 MACE reported in 3 subjects (0.6%): 2 with        myocardial infarction and 1 with stroke.        Conclusions:    -   Efficacy was generally better maintained among Week 28        responders randomized to a fixed interval maintenance dosing        (q12w) compared to those randomized to a subject-tailored        interval maintenance dosing (q12w, q16w, q20, or q24w)        especially for higher level efficacy endpoints (PGA 0, PASI 90,        or PASI 100).    -   No new Safety signals were observed during the study period, and        safety findings were similar between the two randomized groups.    -   Other analyses to better profile subjects in Group 2 who        sustained response with longer dosing intervals through RNA and        DNA analyses are on-going.

Extent of Exposure

A summary of the cumulative dose of ustekinumab received amongrandomized subjects from Week 28 through Week 124 is provided. Theaverage number of study drug administrations was as expected for eachtreatment regimen and time period (6.1 for Group 1 and 4.1 for Group 2).

Primary Endpoint Analysis

Primary Endpoint

The primary endpoint for this study is the number of visits for whichsubjects had a static PGA score of cleared (0) or minimal (1) betweenWeek 88 and Week 112 (the evaluation interval) for subjects randomizedat Week 28 into Groups 1 and 2.

The evaluation interval included a total of 7 visits conducted every 4weeks. The mean (95% confidence interval) number of visits for whichsubjects had a static PGA score of clear (0) or minimal (1) during theevaluation interval was computed for Group 1 and Group 2. Under theassumption of normal distributions, the 95% confidence interval of thedifference in means for the primary endpoint was provided.

The number of visits at which subjects achieved a PGA score of (0) orminimal (1) between Week 88 and Week 112 is summarized in Table 9 below.

Group 2 had fewer visits (mean difference of −0.46) at which subjectshad a PGA score of cleared (0) or minimal (1) during the evaluationinterval compared to Group 1. In addition, Group 1 (55.3%) showed ahigher proportion of subjects with a PGA score of cleared (0) or minimal(1) at all 7 visits during the evaluation interval compared to Group 2(38.1%). During this interval, it is interesting to note that Group 1(55.3%) showed a higher proportion of subjects with a PGA score of 0/1at all 7 visits during the evaluation interval compared to Group 2(38.1%). Further, the proportions of subjects with no visits with a PGAresponse of cleared (0) or minimal (1) during the evaluation intervalwas similar for Group 1 (22.4%) and Group 2 (24.2%).

Subgroup Analysis

In general, there was not a substantial difference in the number ofvisits at which subjects achieved a static PGA score of cleared (0) orminimal (1) (PGA 0/1) during the evaluation interval among variousanalyzed subgroups of Groups 1 and 2. The subgroups are baselinedemographic characteristics, baseline disease characteristics, andpsoriasis medication history. Modest variability was observed in thenumber of visits for which subjects achieve PGA 0/1 between Week 88 andWeek 112 across some subgroups; the observed variability could be due tothe limited sample size within each subgroup.

Major Secondary Endpoint(S) Analysis

Major secondary analyses for the proportions (95% confidence interval)of subjects with a PGA score of cleared (0) or minimal (1) or a PASI 75response over time were based on efficacy evaluable subjects randomizedat Week 28 according to their assigned treatment groups, regardless ofthe actual treatment received. For the major secondary endpoint of thenumber of visits for which subjects achieve a PASI 75 response duringthe Week 88 to Week 112 evaluation interval, the same missing datahandling rules applied to the primary analysis were also used, such thatall randomized subjects were included in the analysis.

PGA Score of Cleared (0) or Minimal (1) Responses from Week 28 ThroughWeek 112 in the Randomized Subjects Population

The PGA responses of cleared (0) or minimal (1) over time from Week 28through Week 112 for Groups 1 and 2 are summarized in FIG. 6. PGA 0/1response rates decreased for both groups during the dose intervaldetermination period (Week 28 through Week 40), (FIG. 6; data on file).As expected, a greater decrease was observed for Group 2 compared toGroup 1, as defining subject-tailored dosing intervals was based onworsening of disease for Group 2 subjects. At Week 40, the proportionsof subjects achieving a PGA 0/1 were 67.1% for Group 1 and 56.4% forGroup 2. After Week 40, some periodicity of responses based onvariations at time points between q12 week injections, as seen in priorustekinumab clinical trials, was observed more notably for Group 1,However, response rates, in general, were maintained at q12 week“trough” visits (e.g., Week 40, 52, 64, etc.), at which the nextinjection of ustekinumab would be due. Response rates for Group 2 werealso generally maintained over time through Week 112. Response rates atmost visits were slightly higher for Group 1 compared to Group 2 (FIG.6).

The Number of Visits for which Subjects Achieved a PASI 75 ResponseBetween Week 88 and Week 112 for Subjects Randomized at Week 28.

The number of visits during the evaluation interval at which subjectshad a PASI 75 response is summarized in Table 10 below. Similar tocorresponding analyses based on PGA responses, Group 2 had slightlyfewer visits at which subjects had a PASI 75 response compared to Groups1 (mean difference of −0.32 visits); on average, Group 1 had 5.8 visitswith PASI 75, and Group 2 had 5.4 visits with PASI 75. Moreover, similarproportions of patients with a PASI 75 response between Groups 1 (69.7%)and Group 2 (66.9%) were observed for each potential number of visitswith a response (ranging from 0 to 7).

Similar results for number of visits at which subjects had a PASI 75response during the evaluation interval were observed for the subgroupanalyses.

The Proportion of Subjects with a PASI 75 Response by Visit from Week 28to Week 112 for Subjects Randomized at Week 28

PASI 75 response rates over time from Week 28 through Week 112 aresummarized in FIG. 7. In general, the pattern of the PASI 75 responsesover time from Week 28 through Week 112 parallels findings for PGA 0/1responses (FIG. 6; data on file). However, the decrease in PASI 75response rates in the Week 28 to Week 40 dose interval determinationperiod is less compared to what was observed for PGA responses in thisinterval. After Week 40, the response rates for Group 1 and Group 2 weregenerally maintained through Week 112. The response rates of Group 1 atmost visits were slightly higher for Group 1 compared to Group 2 fromWeek 44 through week 112. A pattern of q12 week periodicity for PASI 75responses that was more pronounced for Group 1, similar to what wasobserved for PGA 0/1 responses, was noted. Since the PASI measureincorporates both body surface area and qualitative (erythema, scale,induration) elements, it may serve as a more consistent representationof overall disease burden over time compared to PGA alone which accountsonly for qualitative features of psoriasis.

Other Secondary PGA Response Endpoints

PGA Score of Cleared (0) and PGA Score of Mild or Better (≤2) from Week28 Through Week 112 for Subjects Randomized at Week 28

PGA responses of cleared (0) over time from Week 28 through Week 112 aresummarized in FIG. 8. Separation between response curves for Group 1(ustekinumab q12w maintenance regimen) and Group 2 (ustekinumabsubject-tailored interval maintenance regimen) was apparent as early asthe first post randomization visit (Week 32) and the response rates forGroup 1 were consistently higher than those of Group 2 over time.Differences in PGA 0 responses between the two groups were morepronounced than those for either PGA 0/1 or PASI 75 responses,especially at later time points. The PGA scores of mild or better (PGA≤2) over time from Week 28 through Week 112 were comparable for bothgroups and generally stable over time.

PGA Responses from Week 28 Through Week 112 for Subjects in theSubject-Tailored Interval Maintenance Regimen (Group 2)

The PGA scores of cleared (0), PGA scores of cleared (0) or minimal (1),and PGA responses of mild or better (≤2) from Week 28 through Week 112were evaluated for Group 2 (data on file). For PGA responses of cleared(0) and cleared or minimal (0 or 1), as expected, subjects in the Group2 q20 week and q24 week arms performed better than subjects in the Group2 q12 week and q16 week arms. In addition, findings indicate that over25% of initial responders randomized to Group 2 ultimately extendedtheir dosing interval to q24 weeks and that most of these subjectsmaintained response over time.

Other Secondary PASI Response Endpoints

PASI Responses from Week 28 Through Week 112 for Subjects in theSubject-Tailored Interval Maintenance Regimen (Group 2)

The PASI 50, PASI 75, PASI 90 and PASI 100 over time from Week 28through Week 112 for Group 2 are evaluated here (data on file). For PASI75, PASI 90 and PASI 100, as expected, subjects in Group 2's q24w dosingregimen were observed to have better response than subjects in othergroups in Group 2.

The Number of Visits for which Subjects had a PASI 75 Response BetweenWeek 88 and Week 112 for Subjects in the Subject-Tailored IntervalMaintenance Regimen (Group 2)

Results for the number of visits at which Group 2 subjects had a PASI 75response between Week 28 and Week 112 are summarized in Table 11 below.The mean number of visits at which subjects had a PASI 75 response fromWeek 88 through Week 112 increased in parallel with length of dosinginterval.

PASI 90 Responses from Week 28 Through Week 112 for Subjects Randomizedat Week 28

PASI 90 response rates from Week 28 through Week 112 are summarized inFIG. 9 (data on file). Separation between the response curves for Group1 (ustekinumab q12w maintenance regimen) and Group 2 (ustekinumabsubject-tailored interval maintenance regimen) were also observedstarting at the first post randomization visit (Week 32) and maintainedthrough Week 108 (FIG. 9). The proportion of subjects achieving andmaintaining a PASI 90 response was higher for Group 1 compared to Group2. The separation between the PASI 90 response curves for Groups 1 and 2Group 2 is more pronounced compared to findings for PGA 0/1 and PASI 75response curves.

Safety

Safety evaluations focus on the double-blinded portion of the studyextending from Week 28 through Week 124 as comparisons betweenrandomized treatment groups can be made. Supportive data through Week124 are also presented. Table 12 below provides an overview of the keysafety results from 28 through Week 124.

All Adverse Events

For treated subjects randomized at Week 28, from Week 28 through Week124:

-   -   The proportions of subjects experiencing 1 or more AEs were        similar for Group 1 (72.4%) and Group 2 (72.8%); see Table 12        (data on file). AEs were most frequently reported for the        infections and infestations system organ class (SOC) for both        Group 1 (46.1%) and Group 2 (48.7%; 48.1% for Groups 1 and 2        combined).    -   The most common AEs in this SOC were URTI (27.6% in Group 1 and        19.5% in Group 2) and nasopharyngitis (9.2% in Group 1 and 13.2%        in Group 2).        For overall enrolled subjects, from Week 0 through Week 124:    -   Overall, 72.6% of subjects experienced 1 or more AEs. Similarly,        AEs were most commonly reported for the infections and        infestations SOC (49.8%), with URTI (19.9%) and nasopharyngitis        (13.8%) representing the most commonly reported AEs.

Deaths, Other Serious Adverse Events, and Other Significant AdverseEvents

Deaths

There were two deaths through Week 124. One subject (Ser. No.00/009,010) was reported to have died of natural causes. This subjecthad been treated with ustekinumab 90 mg and was randomized to Group 1fixed interval q12 week treatment arm.

The other subject (Ser. No. 00/029,005) died of acute myeloid leukemia(AML). This subject had elevated platelet counts prior to enrollment andAML was diagnosed 2 years after the first study dose. This subject hadbeen receiving ustekinumab 90 mg and was in the Group 2 subject-tailoredq12 week treatment arm.

Other Serious Adverse Events

The proportions of treated subjects randomized at Week 28 whoexperienced one or more serious adverse events (SAEs) through Week 124were low for both Group 1 (9.2%) and Group 2 (7.0%) (Table 5). Noparticular pattern of SAEs was observed and most SAEs were reported assingle and isolated events. Among overall enrolled subjects 8.2%(39/478) experienced 1 or more SAEs from Week 0 through Week 124 (dataon file).

Adverse Events that Resulted in Study Agent Discontinuation

Among treated randomized subjects, the proportions of subjects whodiscontinued study agent between Week 28 and Week 124 due to one or moreAEs (DCAE) were low (6.6% for Group 1, 5.6% for Group 2) [see Table 12].No pattern of AEs leading to discontinuation was observed and most werereported as single events.

Among overall enrolled subjects, 7.3% discontinued study agent due to 1or more AEs between Week 0 and Week 124 (data on file).

Infections, Serious Infections and Infections Requiring Treatment

Among treated randomized subjects, comparable proportions of subjects inGroups 1 and 2 (48.7% and 45.7%, respectively; Table 5) experienced oneor more infections from Week 28 through Week 124. The most commoninfections were URTI (27.6% of subjects in Group 1 and 19.5% in Group 2)and nasopharyngitis (9.2% of subjects in Group 1 and 13.2% in Group 2).

There were no serious infections reported in Group 1, and 3 seriousinfections were reported among Group 2 subjects (Table 12). Reportedserious infections included 1 case of bacterial infection, 1 case ofcystitis, and 1 case of urinary tract infection.

Infections requiring oral or parenteral antimicrobial treatment werereported in 18 of 76 (23.7%) Group 1 subjects and 70 of 302 (23.2%)Group 2 subjects (see Table 12). Overall, URTI was the most frequenttype of infection that required treatment (5.3% in Group 1; 5.0% inGroup 2).

Among overall enrolled subjects, 47.3% experienced 1 or more infectionsbetween Week 0 and Week 124 (data on file). The most commonly reportedinfections were URTI (19.9%) and nasopharyngitis (11.7%). The proportionof subjects with one or more serious infections was 1.5% (data on file).No cases of active TB or opportunistic infection were reported throughWeek 124.

Injection Site Reactions

From Week 28 through Week 124, no injection site reactions (ISRs) werereported among Group 1 subjects for either placebo or 45 mg injections.Among Group 2 subjects, 6 ISRs related to administration of placebo(2.0%) and 1 ISR from an injection of ustekinumab 45 (0.4%) wereobserved. All reported ISRs were of mild severity (data on file).

Possible Anaphylactic or Possible Serum Sickness-Like ReactionsAssociated with Ustekinumab

No subjects experienced a possible anaphylactic reaction or possibleserum sickness-like reaction associated with administration of studyagent through Week 124.

Malignancies

Among treated randomized subjects, 10 reported at least one malignancybetween Week 28 and Week 124 (Table 12). Six of 378 (1.6%) subjects hada non-melanoma skin cancer (NMSC), including 2 of 76 (2.6%) Group 1subjects and 4 of 302 Group 2 subjects (1.3%).

Four of 378 (1.1%) treated randomized subjects reported other types ofmalignancies. One of 76 (1.3%) Group 1 subjects had a transitional cellbladder carcinoma (45 mg dosing). Three of 302 (1.0%) Group 2 subjectshad a malignancy, including 1 case each of pancreatic carcinoma (45 mgdosing), acute myeloid leukemia (90 mg dosing), and chronic myeloidleukemia (90 mg dosing).

Among overall enrolled subjects, malignancies (including NMSC and othermalignancies) 2.5% (12/478) reported a malignancy between Week 0 andWeek 124. Three cases of NMSC and 2 other malignancies (1 colon cancer(90 mg dosing) and 1 prostate cancer (90 mg dosing) were reported inaddition to those described above among treated randomized subjects.

Cardiovascular Events

Two investigator-reported MACE were observed among treated randomizedpatients from Week 28 through Week 124. One Group 2 subject (Subject001050002) receiving ustekinumab 90 mg in the q24 week interval arm hada myocardial infarction (and 1 Group 2 subject (Subject 001027002)receiving ustekinumab 45 mg also in the q24 week interval arm had astroke. One additional investigator-reported MACE, a myocardialinfarction in a subject receiving ustekinumab 45 mg (Subject 001029007),occurred prior to Week 28.

Laboratory Measurements

-   -   Markedly abnormal changes in hematology laboratory values were        observed in some subjects, but rates of markedly abnormal labs        were generally low and comparable between Groups 1 and 2 from        Week 28 through Week 124 (data on file). The most common        reported markedly abnormal change in hematology lab values was        decreased lymphocytes (5.3% [20/378]). Markedly abnormal        hematology lab values occurring on more than 1 occasion were        observed only in Group 2, and included elevated WBC (0.3%),        decreased lymphocyte count (1.3%), and elevated eosinophil count        (0.3%).    -   Markedly abnormal changes in chemistry laboratory values were        also observed in some subjects (data on file). Rates of markedly        abnormal labs were generally low in both Groups 1 and 2. The        only markedly abnormal changes that occurred on more than 1        occasion were elevated alkaline phosphatase, ALT, AST, and total        bilirubin levels, all of which observed in Group 2 subjects and        at a low rate. The most common markedly abnormal change        occurring across both Groups 1 and 2 was elevated ALT level        (3.2% [12/378]).        Immunogenicity

Of the ustekinumab-treated population with evaluable samples throughWeek 124, 63 of 455 (13.8%) patients tested positive for antibodies toustekinumab. This percentage is similar among patients receiving the 45mg dose (n=41; 13.9%) to those receiving the 90 mg dose (n=22; 13.7%)and across Group 1 (n=7; 9.2%) and Group 2 (n=32; 10.6%). A majority ofantibody-positive patients (33 out of 63) had titers ≤1:800. Most of theantibodies developed (from antibody-positive patients with sufficientserum samples) were able to neutralize the bioactivity of ustekinumab invitro (47 out of 62 (75.8%). In testing dosing intervals that alloweddeclining ustekinumab concentrations below quantifiable levels betweeninjections (which approximates multiple withdrawal and retreatmentcycles), no increased susceptibility to anti-drug antibody developed wasidentified. These results suggest that there is no increased risk ofimmunogenicity when increasing the dosing interval to up to a 24-weekinterval.

Predictive Value of PGA=0 at Week 28

Group 2 patients who stably maintained clinical responses over time witha 24-week dosing interval generally demonstrated high levels of responsefor the most stringent measures through the initial run-in treatmentperiod. Because a high proportion of 24-week dosing interval subgrouppatients had a PGA of 0 at Week 28 (Table 6), the utility of thisresponse parameter to be a predictive marker for ability to maintainclinical response at this interval was evaluated. Obtaining a PGA=0 atWeek 28 correlated with a PPV of 60% for maintaining a PGA of 0 or 1with any dosing interval larger than 12 weeks (e.g., 16 weeks, 20 weeks,24 weeks) for ≥five out of seven Week 88 to Week 112 assessment periodvisits. Also, there was a PPV of 44% for maintaining a PGA of 0 or 1with any dosing interval larger than 12 weeks for all seven of thevisits. Obtaining a PGA=0 at Week 28 correlated with PPV's of 44% and32%, respectively, for PGA of 0 for ≥five out of seven Week 88 to Week112 assessment period visits and all seven visits. The sensitivity andspecificity ranged between 61 and 75% for these determinations as shownin Table 6.

TABLE 6 Ability of PGA = 0 at Week 28 to predict long-term response withq12- 24wk response-based dosing, among all patients randomized at Week28 Visits with PGA<2 during Week 88-Week 112 by Sensitivity Specificitymaintenancedosinginterval (TPR)^(a) (FPR)^(b) PPV^(c) NPV^(d) q24wkmaintenance dosing ≥5 visits 75% 72% 44%^(a) 91%^(b)   7 visits 75% 69%32%^(a) 93%^(b) q20wk or q24wk maintenance dosing ≥5 visits 66% 74% 53% 83%    7 visits 69% 70% 41%  88%  >q12wk maintenance dosing^(e) ≥5visits 61% 75% 60%  76%   7 visits 63% 70% 44%  88%  ^(a)Sensitivity, orthe “True Positive Rate” (TPR), represents the proportion of non-q12w orq24w maintenance subgroup patients that can be correctly identified bythe predictive marker or test. ^(b)Specificity, or the “True NegativeRate” (TNR), represents the proportion of patients that should beexcluded from the predicted group that is suggested if the predictivemarker is negative. ^(c)PPV represents the probability of a givenoutcome if a predictive marker is positive, i.e., post-test likelihoodof a positive outcome. As further explanation, these particular PPVs arebased upon the observation that 117/302 Group 2 patients had PGA = 0 atWeek 28 and 51/117 (44%) patients received q24wk maintenance dosing andhad PGA <2 at five or more visits during Week 88-Week 112. Similarly,38/117 (32%) of these Group 2 q24wk patients had PGA <2 at all sevenvisits during Week 88-Week 112. ^(d)NPV represents the probability of anegative outcome if a predictive marker is negative, i.e., post-testlikelihood of a negative outcome. As further explanation, theseparticular NPVs derive from the observation that 17/185 Group 2 patientswith PGA = 1 at Week 28 were treated with q24wk maintenance dosing andhad PGA <2 at five or more visits (NPV = [185 − 17)]/185 = 91%), and13/185 had PGA <2 at all seven visits (NPV = [185 − 13]/185 = 93%)between Week 88-Week 112. ^(e)>q12wk includes q16wk, q20wk, and q24wkmaintenance dosing intervals. FPR—false positive rate, NPV—negativepredictive value, PGA—Physician's Global Assessment, PPV—positivepredictive value, q12/16/20/24wk—every 12/16/20/24 weeks, TPR—truepositive rate

TABLE 7 Schedule for Study Agent Administration Subjects with a staticPGA score of cleared (0) or minimal (1) will be randomizedat Week 28 to1 of 2 Open-label treatment groups. Group 1 will receive run-in theapproved maintenance regimen. period In Group 2, the time of loss ofresponse Double-blind treatment period Weeks (defined as a PGA score ≥2)will (Weeks) 0 4 16 determine the maintenance regimen.^(a) 28^(b) 32^(b)36^(b) 40^(b) 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100 104 A A AGroup Maintenance dosing according to A P P A P A P P A P A P P A P A P1: approved regimen (ustekinumab q12w beginning at Week 28) A A A GroupIf loss of response occurs at 16 P A P P A P A P P A P A P P A P A 2a:weeks after the Week 16 injection (i.e., the Week 32 visit), subjectswill receive ustekinumab q12w beginning at Week 32 A A A Group If lossof response occurs at 20 P P A P P A P P P A P P A P P A P 2b: weeksafter the Week 16 injection (i.e., the Week 36 visit), subjects willreceive ustekinumab q16w beginning at Week 36 A A A Group If loss ofresponse occurs at 24 P P P A P P P A P P P A P P P A P 2c: weeks afterthe Week 16 injection (i.e., the Week 40 visit), subjects will receiveustekinumab q20w beginning at Week 40 A A A Group If no loss of responseoccurs up P P P A P P P P A P P P P A P P P 2d: to 24 weeks after theWeek 16 injection (i.e., the Week 40 visit), subjects will receiveustekinumab q24w beginning at Week 40 A = Active treatment with 45 mgustekinumab (for subjects who weigh ≤100 kg) or 90 mg ustekinumab (forsubjects who weigh >100 kg) P = Placebo  = No study agent administration^(a)Subjects who do not achieve a static PGA of cleared (0) or minimal(1) at Week 28, or have discontinued study agent prior to Week 28, havenot received all 3 study agent injections prior to Week 28 (i.e., theWeek 0, 4, 16 injections), will be discontinued from receiving furtherstudy agent injections, will be followed for safety for at least 20weeks after their last study agent injection, and then will be withdrawnfrom the study. ^(b)At these visits, the PGA score needs to be put intothe IVRS/IWRS. At these visits special consideration should be given tothe timing of the visit to allow for study agent allocation based ondisease activity. Note: At each applicable visit, subjects will receive1 to 2 injections. Those weighing <100 kg will receive 1 injection (45mg ustekinumab or placebo) and those weighing >100 kg will receive 2injections (45 mg ustekinumab + 45 mg ustekinumab or placebo + placebo)depending on treatment group assignment and visit. The dose of studyagent determined at baseline using weight will be the dose utilizedthroughout the study.

TABLE 8 Number of Subjects Who Discontinued Study Agent from Week 28through Week 104; All Subjects Randomized at Week 28 (StudyCNTO1275PSO3009) Group 1 ustekinumab q12w Group 2 ustekinumabsubject-tailored Interval maintenance regimen maintenance All randomizedregimen subjects q12w q16w q20w q24w Total Analysis set: all subjectsrandomized at Week 76  302 84  61  51  84  378 28 Subjects whodiscontinued study agent 17 (22.4%) 67 (22.2%) 17 (20.2%)  7 (11.5%)  7(13.7%) 14 (16.7%) 84 (22.2%) Reason for discontinuation Adverse event 4(5.3%) 15 (5.0%) 4 (4.8%) 3 (4.9%) 2 (3.9%) 3 (3.6%) 19 (5.0%) Death 1(1.3%) 0 0 0 0 0 1 (0.3%) Lack of efficacy 2 (2.6%) 8 (2.6%) 4 (4.8%) 2(3.3%) 1 (2.0%) 1 (1.2%) 10 (2.6%) Lost to follow-Up 6 (7.9%) 12 (4.0%)3 (3.6%) 0 3 (5.9%) 3 (3.6%) 18 (4.8%) Pregnancy 0 3 (1.0%) 1 (1.2%) 0 00 3 (0.8%) Protocol violation 0 4 (1.3%) 1 (1.2%) 0 0 0 4 (1.1%) Studyterminated by sponsor 0 0 0 0 0 0 0 Withdrawal of consent 2 (2.6%) 15(5.0%) 3 (3.6%) 1 (1.6%) 0 6 (7.1%) 17 (4.5%) Other 2 (2.6%) 10 (3.3%) 1(1.2%) 1 (1.6%) 1 (2.0%) 1 (1.2%) 12 (3.2%) [TSIDS01B.RTF][CNTO1275\PSO3009\DBR_WEEK_124\RE_WEEK_124_CSR\PREPROD\TSIDS01B.SAS] 30Sep. 2015, 10:53

TABLE 9 Number of Visits for Which Subjects Achieved a PGA Score ofCleared (0) or Minimal (1) between Week 88 and Week 112; All SubjectsRandomized at Week 28 (Study CNTO1275PSO3009) Group 2 Group 1ustekinumab subject- ustekinumab q12w tailored interval maintenanceregimen maintenance regimen Analysis set: all subjects 76 302 randomizedat Week 28 N^(a) 76 302 Mean (SD) 4.5 (3.07)  4.1 (2.93)  95% confidenceinterval (3.81; 5.21) (3.72; 4.39) of mean^(b) Median 7.0 5.0 Range (0;7) (0; 7) IQ range (1.0; 7.0) (1.0; 7.0) Difference between −0.46treatment groups 95% confidence interval^(b) (−1.20; 0.29) Number ofvisits for which subjects with a PGA score of cleared (0) or minimal (1)0 17 (22.4%)  73 (24.2%) 1 6 (7.9%) 19 (6.3%) 2 2 (2.6%) 17 (5.6%) 3 3(3.9%) 17 (5.6%) 4 2 (2.6%) 16 (5.3%) 5 2 (2.6%) 20 (6.6%) 6 2 (2.6%) 23(7.6%) 7 42 (55.3%) 117 (38.7%) ^(a)After applying treatment failurerules, subjects with any missing data between Weeks 88 and 112 werehandled as follows: (1) If a missing visit was intermittent, the missingvalue was imputed with the weighted average relative to the distance ofthe available values before and after the missing visit (a linearrelationship was assumed across the visits). The resulting score wasrounded to the nearest integer. (2) If a missing visit was notintermittent, i.e., there were no data available after the missingvisit, a last observation carried forward method was used to impute themissing data. ^(b)95% confidence interval was based on normalapproximation.[TEFPGA01A.RTF][CNTO1275\PSO3009\DBR_WEEK_124\RE_WEEK_124_CSR\PROD\TEFPGA01A.SAS]1 Oct. 2015, 14:17

TABLE 10 Group 2 Group 1 ustekinumab subject- ustekinumab q12w tailoredinterval maintenance regimen maintenance regimen Number of Visits forWhich Subjects Achieved a PASI 75 Response between Week 88 and Week 112;All Subjects Randomized at Week 28 (Study CNTO1275PSO3009) Analysis set:all subjects 76 302 randomized at Week 28 N^(a) 76 302 Mean (SD) 5.8(2.31)  5.4 (2.61)  95% confidence interval (5.23; 6.29) (5.15; 5.74) ofmean^(b) Median 7.0 7.0 Range (0; 7) (0; 7) IQ range (6.0; 7.0) (5.0;7.0) Difference between −0.32 treatment groups 95% confidenceinterval^(b) (−0.96; 0.33) Number of visits for which subjects with aPASI 75 response 0 7 (9.2%) 43 (14.2%) 1 2 (2.6%) 7 (2.3%) Number ofVisits for Which Subjects Achieved a PGA Score of Cleared (0) or Minimal(1) between Week 88 and Week 112; All Subjects Randomized at Week 28(Study CNTO1275PSO3009) 2 0  8 (2.6%) 3 5 (6.6%)  5 (1.7%) 4 1 (1.3%)  9(3.0%) 5 2 (2.6%) 11 (3.6%) 6 6 (7.9%) 17 (5.6%) 7 53 (69.7%) 202(66.9%) ^(a)After applying treatment failure rules, subjects with anymissing data between Weeks 88 and 112 were handled as follows: (1) If amissing visit was intermittent, the missing value was imputed with theweighted average relative to the distance of the available values beforeand after the missing visit (a linear relationship was assumed acrossthe visits). (2) If a missing visit was not intermittent, i.e., therewere no data available after the missing visit, a last observationcarried forward method was used to impute the missing data. ^(b)95%confidence interval was based on normal approximation.[TEFPASI01.RTF][CNTO1275\PSO3009\DBR_WEEK_124\RE_WEEK_124_CSR\PROD\TEFPSAI01.SAS]1 Oct. 2015, 14:16

TABLE 11 Number of Visits for Which Subjects Achieved a PASI 75 Responsefrom Week 88 through Week 112; Subjects Randomized to UstekinumabSubject-tailored Interval Maintenance Regimen at Week 28 (StudyCNTO1275PSO3009) Group 2 ustekinumab subject-tailored intervalmaintenance regimen All randomized q12w q16w q20w q24w subjects Analysisset: subjects 84 61 51 84 302 assigned to ustekinumab subject-tailoredinterval maintenance regimen at Week 28 N^(a) 84 61 51 84 302 Mean (SD)4.7 (3.03)  5.2 (2.73)  5.6 (2.37)  6.3 (1.67)  5.4 (2.61)  95%confidence (4.07; 5.38) (4.53; 5.93) (4.92; 6.25) (5.97; 6.70) (5.15;5.74) interval of mean^(b) Median 7.0 7.0 7.0 7.0 7.0 Range (0; 7) (0;7) (0; 7) (0; 7) (0; 7) Number of Visits for Which Subjects Achieved aPGA Score of Cleared (0) or Minimal (1) between Week 88 and Week 112;All Subjects Randomized at Week 28 (Study CNTO1275PSO3009) Group 2ustekinumab subject- Group 1 ustekinumab q12w tailored intervalmaintenance maintenance regimen regimen IQ range (1.0; 7.0) (3.0; 7.0)(5.0; 7.0) (7.0; 7.0) (5.0; 7.0) Number of visits for which subjectswith a PASI 75 response 0 20 (23.8%)  9 (14.8%) 5 (9.8%) 3 (3.6%) 43(14.2%) 1 3 (3.6%) 1 (1.6%) 2 (3.9%) 1 (1.2%) 7 (2.3%) 2 3 (3.6%) 4(6.6%) 1 (2.0%) 0 8 (2.6%) 3 0 2 (3.3%) 0 3 (3.6%) 5 (1.7%) 4 2 (2.4%) 3(4.9%) 2 (3.9%) 2 (2.4%) 9 (3.0%) 5 2 (2.4%) 0 4 (7.8%) 5 (6.0%) 11(3.6%)  6 8 (9.5%) 2 (3.3%)  6 (11.8%) 1 (1.2%) 17 (5.6%)  7 46 (54.8%)40 (65.6%) 31 (60.8%) 69 (82.1%) 202 (66.9%)  ^(a)After applyingtreatment failure rules, subjects with any missing data between Weeks 88and 112 were handled as follows: (1) If a missing visit wasintermittent, the missing value was imputed with the weighted averagerelative to the distance of the available values before and after themissing visit (a linear relationship is assumed across the visits). (2)If a missing visit was not intermittent, i.e., there were no dataavailable after the missing visit, a last observation carried forwardmethod was used to impute the missing data. ^(b)95% confidence intervalwas based on normal approximation.[TEFPASI04.RTF][CNTO1275\PSO3009\DBR_WEEK_124\RE_WEEK_124_CSR\PROD\TEFPASI04.SAS]1 Oct. 2015, 14:16

TABLE 12 Summary of Key Safety Findings from Week 28 Through Week 124;Treated Subjects Randomized at Week 28 (Study CNTO1275PSO3009) Group 1ustekinumab q12w Group 2 ustekinumab subject-tailored intervalmaintenance regimen maintenance All randomized regimen subjects q12wq16w q20w q24w Total Analysis set: treated subjects randomized at 76 30284 61 51 84 378 Week 28 Avg duration of follow-up (wks) 85.9 84.2 87.289.9 90.1 90.9 84.6 Avg exposure (number of administrations) 14.6 14.615.3 15.7 15.7 15.9 14.6 for injections at or after Week 28 Subjects whodiscontinued study agent 5 (6.6%) 17 (5.6%) 5 (6.0%) 3 (4.9%) 2 (3.9%) 3(3.6%) 22 (5.8%) because of 1 or more adverse events Subjects with 1 ormore: Adverse events 55 (72.4%) 220 (72.8%) 62 (73.8%) 54 (88.5%) 35(68.6%) 62 (73.8%) 275 (72.8%) Serious adverse events 7 (9.2%) 21 (7.0%)6 (7.1%) 2 (3.3%) 2 (3.9%) 9 (10.7%) 28 (7.4%) Overall infections 37(48.7%) 138 (45.7%) 45 (53.6%) 38 (62.3%) 19 (37.3%) 35 (41.7%) 175(46.3%) Serious infections 0 3 (1.0%) 2 (2.4%) 0 1 (2.0%) 0 3 (0.8%)Requiring treatment 18 (23.7%) 70 (23.2%) 22 (26.2%) 20 (32.8%) 12(23.5%) 15 (17.9%) 88 (23.3%) Malignancy 3 (3.9%) 7 (2.3%) 3 (3.6%) 1(1.6%) 1 (2.0%) 1 (1.2%) 10 (2.6%) MACE* 0 2 (0.7%) 0 0 0 2 (2.4%) 2(0.5%) *MACE: myocardial infarction (MI), stroke or CV deathSequences

SEQ ID NO: 1 <211> 5 <212> PRT <213> Homo sapiens <400> 1Thr Tyr Trp Leu Gly 1              5 SEQ ID NO: 2 <211> 17 <212> PRT<213> Homo sapiens <400> 2Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe Gln1               5                   10                  15 GlySEQ ID NO: 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe1               5                   10 SEQ ID NO: 4 <211> 11 <212> PRT<213> Homo sapiens <400> 4 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1               5                   10 SEQ ID NO: 5 <211> 7 <212> PRT<213> Homo sapiens <400> 5 Ala Ala Ser Ser Leu Gln Ser 1               5SEQ ID NO: 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6Gln Gln Tyr Asn Ile Tyr Pro Tyr Thr 1               5 SEQ ID NO: 7 <211>119 <212> PRT <213> Homo sapiens <400> 7Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1               5                   10                  15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr            20                  25                  30Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile        35                  40                  45Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe    50                  55                  60Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr65                  70                  75                  80Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                85                  90                  95Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly            100                 105                110Thr Leu Val Thr Val Ser Ser         115 SEQ ID NO: 8 <211> 108 <212> PRT<213> Homo sapiens <400> 8Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1               5                   10                  15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp            20                  25                  30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile        35                  40                  45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly    50                  55                  60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65                  70                  75                  80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr                85                  90                  95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg            100                 105 SEQ ID NO: 9 <211> 503 <212> PRT<213> Homo sapiens <400> 9Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu1               5                   10                  15His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys            20                  25                  30Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp        35                  40                  45His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu    50                  55                  60Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr65                  70                  75                  80Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe                85                  90                  95Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr            100                 105                 110Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys        115                 120                 125Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu    130                 135                140Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser145                 150                 155                160Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu               165                  170                 175Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser            180                 185                 190Tyr Leu Asn Ala Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val        195                 200                 205Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr    210                 215                 220Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser225                 230                 235                 240Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu                245                 250                 255Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu            260                 265                 270Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser        275                 280                 285Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu    290                 295                 300Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu305                 310                 315                 320Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly                325                 330                 335Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala            340                 345                 350Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys        355                 360                 365Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu    370                 375                 380Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser385                 390                 395                 400Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu                405                 410                 415Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu            420                 425                 430Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe        435                 440                 445Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val    450                 455                 460Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser465                 470                 475                 480Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu                485                 490                 495Trp Ala Ser Val Pro Cys Ser             500

What is claimed is:
 1. A method of treating psoriatic arthritis in a patient using an increasing dosing or maintenance interval, comprising administering a pharmaceutical composition comprising an antibody to both IL-12 and IL-23 to the patient, wherein the antibody comprises a heavy chain variable amino acid sequence of SEQ ID NO: 7 and a light chain variable amino acid sequence of SEQ ID NO: 8, in an initial dose, a dose 4 weeks after the initial dose and a dose once every 12 weeks for 24 weeks after administration of the initial dose and increasing the dosing interval 28 weeks after administration of the initial dose to a dosing interval of every 24 weeks after identifying the patient as a responder to the antibody 28 weeks after administration of the initial dose, wherein the dose is 45 mg or 90 mg.
 2. The method of claim 1, wherein the antibody to IL-12 and IL-23 administered to the patient is ustekinumab.
 3. The method of claim 1, wherein the patient does not show an increased risk of immunogenicity.
 4. The method of claim 1, wherein the antibody comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence of SEQ ID NO: 8 and comprises about 0.53 mg L-histidine per ml of the pharmaceutical composition; about 1.37 mg L-histidine monohydrochloride monohydrate per ml of the pharmaceutical composition; about 0.04 mg polysorbate 80 per ml of the pharmaceutical composition; about 76 mg of sucrose per ml of the pharmaceutical composition; and water as a diluent at standard state.
 5. The method of claim 4, wherein the antibody is ustekinumab.
 6. A method of treating psoriatic arthritis in a patient using an increasing dosing or maintenance interval, comprising administering a pharmaceutical composition comprising an antibody to both IL-12 and IL-23 to the patient in an initial dose, a dose 4 weeks after the initial dose and a dose once every 12 weeks for 24 weeks after administration of the initial dose and increasing the dosing interval 28 weeks after administration of the initial dose to a dosing interval of every 24 weeks after identifying the patient as a responder to the antibody 28 weeks after administration of the initial dose, wherein the dose is 45 mg or 90 mg, and wherein the antibody comprises the heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and the light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 and comprises about 0.53 mg L-histidine per ml of the pharmaceutical composition; about 1.37 mg L-histidine monohydrochloride monohydrate per ml of the pharmaceutical composition; about 0.04 mg polysorbate 80 per ml of the pharmaceutical composition; about 76 mg of sucrose per ml of the pharmaceutical composition; and water as a diluent at standard state. 